Cloning and expression of a novel acetylcholine-gated ion channel receptor subunit

ABSTRACT

The present invention provides isolated nucleic acids encoding alpha9 nicotinic acetylcholine receptor subunit and receptor subunit protein encoded thereby. Also provided are vectors containing the invention nucleic acids, host cells transformed therewith, alpha9 nicotinic acetylcholine receptor subunit and functional nicotinic acetylcholine receptors comprising at least one alpha9 subunit expressed recombinantly in such host cells as well as transgenic non-human mammals that express the invention receptor subunit and mutants thereof. Receptors of the invention comprise at least one alpha9 nicotinic acetylcholine subunit and form cationic channels activated by acetylcholine, but blocked by nicotine and muscarine. The invention also provides methods for identifying compounds that modulate the ion channel activity of the functional invention receptors containing at least one invention subunit.

This invention was made with Government support under Grant NumberNS-11549, awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

RELATED INVENTIONS

This invention is related to U.S. Ser. No. 07/898,185, filed Jun. 12,1992, now U.S. Pat. No. 5,371,188, which is a continuation of U.S. Ser.No. 07/664,473, filed Mar. 4, 1991, now abandoned, which is, in turn, acontinuation of U.S. Ser. No. 07/321,384, filed Mar. 14, 1989, nowabandoned, which is, in turn, a continuation-in-part of U.S. Ser. No.07/170,295, filed Mar. 18, 1988, now abandoned, all of which are herebyincorporated by reference herein in their entirety.

RELATED INVENTIONS

This invention is related to U.S. Ser. No. 07/898,185, filed Jun. 12,1992, now U.S. Pat. No. 5,371,188, which is a continuation of U.S. Ser.No. 07/664,473, filed Mar. 4, 1991, now abandoned, which is, in turn, acontinuation of U.S. Ser. No. 07/321,384, filed Mar. 14, 1989, nowabandoned, which is, in turn, a continuation-in-part of U.S. Ser. No.07/170,295, filed Mar. 18, 1988, now abandoned, all of which are herebyincorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

Intercellular communication is essential for the function ofmulticellular systems. Ion channel proteins, as mediators of informationtransfer in the brain, endocrine system, enteric nervous system andneuromuscular junction, modulate ion fluxes that produce voltage changesacross cell membranes and simultaneously act as sensors of physiologicalsignals, for example, changes in ligand concentrations and intransmembrane voltage. Ligand-gated ion channels provide for rapiddialogue between cells of the central nervous system, converting achemical neurotransmitter signal released from one cell into anelectrical signal that propagates along the cell membrane of a targetcell. Ligand-gated ion channels are multimeric protein complexes withcomponent subunits encoded by related genes.

At the present time, numerous families of ligand-gated receptors havebeen identified and characterized on the basis of sequence identity.Those which form cationic channels include, for example, excitatorynicotinic acetylcholine receptors (nAChRs), excitatoryglutamate-activated receptors, the 5-HT₃ serotonin receptor, the ATPreceptor and the sarcoplasmic ryanodine receptor. Those which formanionic channels include, for example, the inhibitory GABA andglycine-activated receptors.

The neurotransmitter acetylcholine (ACh) activates two pharmacologicallydifferent receptor types: nicotinic acetylcholine receptors (nAChR) fromthe ligand-gated ion channel superfamily and muscarinic acetylcholinereceptors (mAChR) from the G-protein coupled receptor superfamily(Taylor, A. Goodman-Gilman, T. H. Rall, A. S. Nies and P. Taylor, eds.(New York:Pergamon Press), pp. 166-186, 1990); (Taylor, A.Goodman-Gilman, T. H. Rall, A. S. Nies and P. Taylor, eds. (NewYork:Pergamon Press), pp. 122-149, 1990). A number of pathologies and/ordisease conditions are associated with nAChRs, such as, for example,myasthenia gravis, schizophrenia, Alzheimer's disease, Tourette'sdisease and nicotine addiction. Biochemical and electrophysiologicaldata have shown that nicotinic and muscarinic receptors are functionallydistinct entities. (Bonner, et al., Science, 237,527-532, 1987). WhereasnAChRs are pentamers composed of related protein subunits that span theplasma membrane four times, mAChRs are formed by a single polypeptidechain which is postulated to span the plasma membrane seven times.

Nicotinic acetylcholine receptors, glycoproteins composed of fivesubunits, transduce the binding of acetylcholine in the cationicchannel. The five receptor subunits form a pseudosymmetric ring around acentral channel. Neuronal nicotinic AChRs (NnAChRs) mediateneurotransmission at many central and peripheral synapses, and comprisetwo subunit types (alpha and beta) encoded by 10 different neuronalgenes. Expression of particular combinations of subunit RNAs in oocytesyields biophysically distinct channels that are distinguishedpharmacologically on the basis of ligands that modulate such channels.

Recombinant DNA technology has enabled the identification of thevertebrate muscle nAChR subunits alphal, betal, gamma, delta and epsilonand the neuronal subunits alpha2, alpha3, alpha4, alpha5, alpha6,alpha7, alpha8, beta2, beta3 and beta4 (rat nomenclature). Variouscombinations of these subunits produce functional recombinantreceptor-channel complexes that are activated by both ACh and nicotine.The nAChR at the neuromuscular junction is thought to have a (α1)₂ β1γδstoichiometry (Galzi, et al., Annu. Rev. Pharmacol., 31, 37-72, 1991).In contrast, the neuronal nAChR subunits alpha2, alpha3 and alpha4 leadto the assembly of functional nAChRs in concert with either beta2 orbeta4 (Boulter, et al. Proc. Natl. Acad. Sci. USA, 84, 7763-7767, 1987;Ballivet, et al., Neuron, 1, 847-852, 1988; Wada, et al., Science, 240,330-334, 1988; Deneris, et al., Neuron, 1, 45-54, 1988; Duvoisin, etal., Neuron, 3, 487-496, 1989; Couturier, et al., J. Biol. Chem, 265,17560-17567, 1990), while the neuronal alpha7 and alpha8 subunits canform functional nAChRs in the absence of any other subunit (Couturier,et al., J. Biol. Chem, 265, 17560-17567, 1990; Seguela, et al., J.Neurosci, 13, 596-604, 1993; Gerzanich, et al., Molec. Pharmacol., 45,212-220, 1994).

Given the existence of ten distinct nicotinic acetylcholine subunitgenes, numerous combinations of subunits producing functional receptorsare possible. In spite of the numerous combinations of subunits whichcan be prepared from previously cloned genes, the properties of thenative nAChRs do not always match those of recombinant receptors(Sargent, Annu. Rev. Neurosci., 16, 403-443, 1993). For example, thecholinergic receptors present in bovine chromaffin cells and in rat andchick cochlear hair cells exhibit a pharmacological profile that doesnot fit any combination of known subunits (Shirvan, et al., Proc. Natl.Acad. Sci. USA., 88, 4860-4864, 1991; Housley, et al., Proc. R. Soc.Lond. B, 244, 161-167, 1991; Fuchs, et al., Proc. R. Soc. Lond. B, 248,35-40, 1992; Erostegui, et al., Hearing Res., 74, 135-147, 1994), thussuggesting the existence of additional, as yet unidentified subunits.

Thus, a need exists for identifying additional members of the nicotinicacetylcholine receptor superfamily, and characterizing such nAChRsubunits, as well as functional receptors assembled therefrom, whichincludes elucidation of the nature of assembly of various subunits inthe production of a functional receptor (i.e., a subunit assemblycontaining ligand binding sites and a ligand-gated transmembranechannel), and the relationship between the structure of the subunitassembly and the pharmacological profile of the corresponding receptor.The present invention satisfies these needs and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acids encoding alpha9nicotinic acetylcholine receptor (nAChR) subunit, isolated receptorsubunit protein encoded thereby as well as recombinately expressedalpha9 nicotinic acetylcholine receptor (nAChR). Further provided arevectors and probes containing such nucleic acids, host cells transformedwith such nucleic acids, antisense oligonucleotides and compositionscontaining such oligonucleotides, antibodies that specifically bind toinvention receptors and compositions containing such antibodies as wellas transgenic non-human mammals.

The alpha9 nAChR subunits of the invention form a cationic receptorchannel complex which is activated by acetylcholine and is permeable tocations, including calcium. Functional alpha9 nACh receptors of theinvention may be expressed as homomeric receptors, i.e., only one typeof subunit is required for function, or invention receptors may beexpressed as heteromeric receptors wherein more than one type of subunitis required to form a functional receptor. Additionally, the inventionprovides methods for identifying compounds that modulate activity of theinvention receptors, or the activity of nucleic acid encoding suchreceptors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide and the amino acid sequence of the cDNAclone coding for the alpha9 nAChR subunit. The amino acid sequence isshown below the nucleotide sequence. Cleavage of the signal peptide ispredicted at amino acid position 1 (Von-Heijne, Nucl. Acid. Res., 14,4683-4691, 1986). Amino acids encoding the signal peptide are assignednegative numbers. Nucleotides are numbered in the 5' to 3' direction,starting with the first nucleotide of the codon for the putativeN-terminal residue of the mature protein. Nucleotides on the 5' side ofamino acid residue 1 are indicated by negative numbers. Arrowheadsindicate the intron location determined by genomic sequencing. Membranespanning regions are underlined. The sequence information presented inFIG. 1 is also presented in Patentin format in SEQ ID NOs 1 and 2.

FIG. 2A shows the restriction map for the alpha9 subunit gene, and FIG.2B shows a partial restriction map for overlapping genomic clones, M6and MNANO, spanning the entire coding sequence of the alpha9 subunitgene. NcoI and NheI restriction sites not mapped in pMNANO, and SacIrestriction sites are not mapped in pM6.

FIG. 3 shows the alignment of amino acid sequences for known nAChR alphasubunits. All sequences correspond to rat subunits, except for alpha8which is a chick subunit. Identical residues in all sequences arepresented as white letters in a black background. Spaces are introducedto maximize homologies. Predicted signal peptides and the four potentialmembrane spanning regions are indicated. Asterisks denote cysteineresidues 127, 141, 191 and 192 (alpha9 numbering for the mature peptide,absent the 28 amino acid residues comprising the signal peptide)conserved in all nAChR alpha subunits. The sequence informationpresented in FIG. 3 is also presented in Patentin format n SEQ ID NOs2-8.

FIGS. 4A and 4B show electrophysiological responses of alpha9 injectedoocytes to cholinergic agonists. FIG. 4A shows current responseselicited by ACh, nicotine, muscarine, 1,1-dimethyl-4-phenylpiperazinium(DMPP), and oxotremorine-M (OXO-M) in oocytes injected with alpha9 cRNAand held at -50 mV under voltage-clamp.

FIG. 4B shows concentration-response curves to ACh, DMPP and OXO-M.Values represented are the mean and standard error of the mean of peakcurrent values obtained in at least four oocytes per drug. Error barsare not shown when the standard error is smaller than the symbol.Responses from each cell were normalized to the maximal current evokedby ACh. The Hill equation (EC₅₀ =9.7 μM; slope=1.3) was fitted to theACh concentration-responsive curve.

FIGS. 5A and 5B show the blockage of ACh responses in alpha9 injectedoocytes by various antagonists. Inhibition curves were performed by thecoapplication of 10 μM ACh and increasing concentrations of either (-)nicotine or (+) muscarine (see FIG. 5A) and strychnine, d-tubocurarine(d-TC) or atropine (see FIG. 5B). Responses are expressed as thepercentage (%) of the control current evoked by 10 μM ACh. The mean andstandard error of the mean of values obtained in at least four differentoocytes per drug are shown. Error bars are not shown when the standarderror is smaller than the symbol.

FIG. 6 shows the sensitivity of ACh-evoked currents in alpha9 injectedoocytes to α- and κ-bungarotoxin. Representative current responses to100 μM ACh recorded at a holding potential of -50 mV are shown. Oocyteswere preincubated with α-bungarotoxin (α-BTX, A) or κ-bungarotoxin(κ-BTX, B) for 30 minutes before the application of the second testconcentration of ACh.

FIGS. 7A through 7C show the voltage-dependence of the ACh-evokedcurrents in alpha9 injected oocytes and Ca²⁺ permeability of therecombinant alpha9 receptor. In FIG. 7A the current-voltage relationshipof ACh-evoked currents in alpha9-injected oocytes was determined byapplying a voltage ramp (2 second duration, +50 mV to -120 mV) duringthe plateau phase of the current response. The traces are representativeof those obtained in four different oocytes.

FIG. 7B shows representative current traces elicited by 100 μM ACh inalpha9-expressing oocytes before and after the injection of 50 nl of 20mM 1,2-bis(2-aminophenoxy)ethane-N,N,N¹,N¹ -tetraacetic acid (BAPTA).

FIG. 7C shows ACh-evoked currents in alpha9-injected oocytes held at -10mV under voltage-clamp and bathed with a Ringer's solution containing350 mM NaCl.

FIG. 8 shows detection of alpha9 transcripts in the rat cochlea.Amplification reactions were performed as described in Example VI, usingcDNA transcribed from total RNA as template and alpha9 specific primers.Resolution of the amplified products in a 1.5% agarose gel stained withethidium bromide is shown. An aliquot of 10 μM of each reaction mixturewas loaded per lane. Lane 1, DNA ladder; lane 2, no DNA template; lane3, amplified product from olfactory epithelium cDNA; lane 4, amplifiedproduct from olfactory epithelium cDNA; lane 5, amplified product fromsciatic nerve cDNA.

FIGS. 9A through 9F show the results of in situ hybridization ofsagittal sections of rat embryos and coronal sections of adult brainsand identification and transcript localization of alpha9. FIGS. 9A and9B show the presence of alpha9 transcripts in the hypophyseal gland, theolfactory epithelium, the sternohyoid muscle and the tongue, of a ratembryo at stage E16. FIGS. 9C and 9D show a high magnification view ofthe pituitary in a rat embryo at stage E16 where alpha9 transcript islocated in the pars tuberalis but not the pars distalis or the parsnervosa. FIGS. 9E and 9F show the presence of alpha9 transcripts in thepars tuberalis of the adult rat brain.

DETAILED DESCRIPTION OF THE INVENTION

Molecular cloning studies have demonstrated structural and functionaldiversity in nicotinic acetylcholine receptors (nAChRs). To date, sevenalpha subunits (alpha2 to alpha8) and three beta subunits (beta2 tobeta4) have been characterized in the nervous system of vertebrates. Thepresent invention describes the identification and functionalcharacterization of a new member of this family of receptor subunitgenes that are activated by the neurotransmitter acetylcholine (ACh).The new member is designated alpha9. The molecular structure of alpha9indicates that it belongs to the ionotropic (nicotinic) rather than tothe metabotropic (muscarinic) ACh receptor family. However, the mixednicotinic-muscarinic properties of the recombinant alpha9 receptordiffer from the pharmacological profile of all known functionalnicotinic receptors.

Isolation and identification of the novel nAChR subunit gene of thepresent invention was accomplished by screening a rat genomic libraryusing a rat nAChR alpha7 subunit cDNA as probe. DNA sequence analysisrevealed that one isolated genomic clone encoded a protein withsignificant amino acid sequence identity with members of theligand-gated ion channel gene superfamily. Its homology to knownsubunits revealed that it was more related to nAChR subunits than toGABA_(A), glycine or 5-HT₃ receptor subunits. The presence of conservedcontiguous cysteine residues in the extracellular domain, which are ahallmark of all nAChR alpha subunits and are thought to be part of theacetylcholine binding domain (Popot and Changeux, Physiol. Rev. 64,1162-1193, 1984) suggested that this gene encoded a nAChR alpha subunit.Therefore, in accordance with current nomenclature, this newlydiscovered subunit has been designated the alpha9 subunit of the nAChRgene family.

A polymerase chain reaction (PCR) fragment derived from the isolatedgenomic clone was used to screen a rat olfactory epithelium cDNAlibrary. Four independent cDNA clones were isolated, one of whichcontained a 1937 bp insert encoding an open reading frame for the alpha9subunit. The nucleotide and deduced amino acid sequences are shown inFIG. 1 (and are also presented in Patentin format in SEQ ID NOs 1 and2). The full length alpha9 cDNA encodes a mature protein of 451 aminoacid residues, preceded by a leader sequence of 28 residues. It containsall the features characteristic of other members of the nAChR genefamily, including four hydrophobic regions which predict potentialmembrane spanning regions, MSR I to IV (Kyte and Doolitle, J. Mol.Biol., 157, 105-132, 1982), and cysteine residues at positions 127, 141,191 and 192 (alpha9 numbering for the mature peptide, absent the 28amino acid residues comprising the signal peptide) which are present inall nAChR alpha subunits.

The full-length alpha9 cDNA was used as a probe to screen two mousegenomic libraries constructed in phage vectors lambdaDASH II andlambdaFIX II. Two overlapping genomic clones were obtained (see FIG. 2).These clones, spanning the entire coding sequence of the alpha9 subunitgene, were cloned into plasmid vectors and the alpha9 subunit genestructure was determined by sequencing across the intron-exonboundaries. The intron-exon boundaries of the alpha9 gene are indicatedin FIG. 1. The gene consists of five exons and has an intron-exonstructure that differs from that of all known nAChR genes (Noda, et al.,Nature, 305, 818-823, 1983; Nef, et al., EMBO J., 7, 595-601, 1988;Wada, et al., Science, 240, 330-334, 1988; Buonanno, et al., J. Biol.Chem., 264, 7611-7616, 1989; Boulter, et al., J. Biol. Chem, 265,4472-4482, 1990). For example, in contrast to other nAChR subunit geneswhere the intron-exon boundaries of the first four exons are conserved,exons III and IV of the alpha9 gene are fused.

The alpha9 cDNA clone was sequenced and the sequence compared withsequences of other nAChR alpha subunits (see FIG. 3). Based on sequencesimilarity, the alpha9 subunit appears to be a distant member of thenAChR subunit gene family. It is as distinct from the neuronalalpha7-alpha8 subfamily (38% amino acid sequence identity) as it is fromthe neuronal alpha2-alpha6 (36-39%) subfamily or from the muscle alphalsubunit (37%). Although alpha9 shares the most highly conserved sequenceelements with other members of the family, some amino acid residuesdiffer from those found invariant in the other alpha subunits. Forexample, the conserved hydrophobic residues Phe-99 and Val-230 (alpha9numbering for the mature peptide, absent the 28 amino acid residuescomprising the signal peptide) are changed to the polar residues Ser-99and Ser-230 in the alpha9 protein and the conserved positively chargedresidue Lys-144 is substituted by the non-charged residue Thr-144. Thehydrophobic residues Leu-255 (alpha1-alpha6 subunits) or Met-255(alpha7-alpha8 subunits) present in MSR II, are replaced by the polaramino acid Gln-255 in the alpha9 subunit. In addition, when compared toother nAChR subunits, alpha9 has a deletion of a Thr residue between MSRII and MSR III.

A full-length alpha9 cDNA suitable for Xenopus oocyte expression studieswas constructed by subcloning the fragment from nucleotide -94 to 1766(FIG. 1; i.e., residues 79 to 1938 as presented in SEQ ID NO:1) into theexpression vector pGEMHE (Liman et al., Neuron, 9, 861-871, 1992). cRNAwas synthesized using the mMessage mMachine transcription kit (Ambion,Austin, Tx.), with plasmid linearized with NheI.

Two days after the injection of alpha9 cRNA, more than 95% ofvoltage-clamped Xenopus oocytes responded to acetylcholine. Inwardcurrents in response to 100 μM acetylcholine ranged from 20 to 500 nA.FIG. 4A shows representative current traces in response to theapplication of acetylcholine. High concentrations (>10 μM) of thisagonist evoked a fast peak response which rapidly decayed to a plateaulevel. Oocytes expressing alpha9 were insensitive to glutamate, GABA,glycine, serotonin, ATP, histamine and adenosine.

All functional nAChR alpha subunits cloned prior to the cloning of thealpha9 subunit, upon expression in Xenopus oocytes, form eitherheteromeric or homomeric receptor-channel complexes activated bynicotine (Boulter et al., Proc. Natl. Acad. Sci. USA, 84, 7763-7767,1987; Duvoisin et al., Neuron, 3, 487-496, 1989; Couturier et al.,Neuron, 5, 847-856, 1990; Luetje and Patrick, J. Neurosci. 11, 837-845,1991; Seguela et al., J. Neurosci., 13, 596-604, 1993; Gerzanich et al.,Molec. Pharmacol., 45, 212-220, 1994). Strikingly, nicotine (0.1 μM to 1mM) did not elicit any response in alpha9-injected oocytes (FIG. 4A).Co-expression of alpha9 with either beta2 or beta4 nAChR subunits didnot result in the formation of receptor-channels that were activated bynicotine. The alpha9 receptor-channel complex was also not activated bymuscarine (FIG. 4A). Moreover, neither the nicotinic agonist cytosinenor the muscarinic agonists bethanecol and pilocarpine elicited currentresponses. However, both the nicotinic agonist1,1-dimethyl-4-phenylpiperazinium (DMPP) and the muscarinic M1 agonistoxotremorine M (OXO-M), induced inward currents in alpha9-injectedoocytes (FIG. 4A). FIG. 4B shows the concentration-response curves tothese cholinergic agonists. Acetylcholine had an apparent affinity(EC₅₀) of 10 μM. The maximum current responses elicited by both DMPP andOXO-M were approximately 5% of that observed with acetylcholine.

Although neither nicotine nor muscarine evoked responses in oocytesinjected with alpha9 cRNA (see FIG. 4A), both of these classiccholinergic agonists reduced the currents evoked by acetylcholine. FIG.5A shows the inhibition curves that resulted from the co-application of10 μM acetylcholine with increasing concentrations of either nicotine ormuscarine (IC₅₀ =30 μM and 75 μM, respectively). As shown in FIG. 5B,the alpha9 receptor-channel complex was also blocked by the nicotinicantagonist d-tubocurarine (IC₅₀ =0.3 μM), as well as by the muscarinicantagonist atropine (IC₅₀ =1.3 μM). The alkaloid strychnine, classicallyused as a blocker of glycine-gated chloride channels, was found to be apotent antagonist of alpha9 homomers, with an IC₅₀ of 0.02 μM (FIG. 5B).Both α-bungarotoxin (100 nM) and κ-bungarotoxin (100 nM) blockedresponses to 100 μM acetylcholine (FIG. 6). The blockade by these toxinswas almost completely reversed after a 10 minute wash of the oocyteswith frog Ringer's solution.

Electrophysiological properties were determined on alpha9 injectedoocytes 2-7 days after injection. The current-voltage (I-V) relationshipobtained by the application of a 2 second voltage ramp at the plateauresponse to acetylcholine is shown in FIG. 7A. The I-V curve wasnon-linear, displaying a maximal inward current elicited byacetylcholine at -50 mV. Current responses were reduced at potentialsnegative to -50 mV. The fact that the ratio between the inward currentelicited by 100 μM acetylcholine and that evoked by 1 μM acetylcholinewas greater at -50 mV (2.1) than at -80 mV (1.0), indicates that thereduction in current responses at hyperpolarized potentials may dependupon agonist concentration. At holding potentials more positive than -50mV, the inward currents activated by acetylcholine decreased until -25mV, where a strong rectification was observed up to a holding potentialof +20 mV. I-V curves for both peak and plateau responses performed withstepwise increments in the holding potential, had the same shape asshown in FIG. 7A.

From the I-V relationships, an apparent reversal potential of -25 mV isestimated. This value is compatible with either a non-selective cationiccurrent or with an anionic (Cl⁻) current. The change in external NaClconcentration from 50 mM to 150 mM produced a positive shift in thereversal potential of acetylcholine-induced currents. This indicatesthat the alpha9 channel is permeable to Na⁺. Most of the peak responseelicited by 100 mM acetylcholine in alpha9-expressing oocytesdisappeared when oocytes were injected with the calcium chelator1,2-bis(2-aminophenoxy) ethane-N,N,N¹,N¹ -tetraacetic acid (BAPTA) (seeFIG. 7B). Thus, as has been suggested for other nAChR subunits(Gerzanich et al., Molec. Pharmacol., 45, 212-220, 1994), this resultindicates that part of the current evoked by acetylcholine is carried bya Cl⁻ current through Ca²⁺ activated Cl⁻ channels known to be present inoocytes (Miledi and Parker, J. Physiol. (Lond)., 357, 173-183, 1984). Inorder to further test the participation of a Ca²⁺ activated Cl⁻ currentin response to acetylcholine, the reversal potentials of Cl⁻ and Na⁺were shifted in opposite directions by transiently raising the externalNaCl concentration to 350 mM and holding the oocytes at -10 mV under twoelectrode voltage clamp. Under this condition, 100 μM acetylcholineelicited both an outward current followed by an inward current (FIG.7C). As reported for other neuronal nAChRs (Vernino et al., Neuron, 8,127-134, 1992; Seguela et al., J. Nerousci., 12, 596-604, 1993), theinward current probably results from the influx of cations throughalpha9 receptor-channels and the outward current from the flux of Cl⁻through Ca²⁺ -activated Cl⁻ channels. It should be noted that I-V curvesperformed in 1,2-bis(2-aminophenoxy)ethane N,N,N¹,N¹ -tetraacetic acidinjected oocytes had the same shape as that described above, suggestingthat the Cl⁻ current did not contribute to the I/V curve under theconditions of the experiment.

The above-described Xenopus oocyte expression studies demonstrate thatthe alpha9 protein subunit forms ion channels activated by acetylcholineand permeable to both Na⁺ and Ca²⁺. Similar to the alpha7 and alpha8neuronal subunits (Couturier et al., Neuron, 5, 847-856, 1990; Gerzanichet al., Molec. Pharmacol., 45, 212-220, 1994), alpha9 can assemble intoa homomeric receptor-channel complex. This differs from other functionalneuronal nAChR alpha subunits which require co-assembly with a betasubunit in order to form receptor-channel complexes (Boulter et al.,Proc. Natl. Acad. Sci. USA, 84, 7763-7767, 1987; Ballivet et al.,Neuron, 1, 847-852, 1988; Wada et al., Science, 240, 330-334, 1988).

Currents elicited by acetylcholine in alpha9-injected oocytes decreasedat holding potentials negative to -50 mV. This could result from avoltage-dependent blockade of the channel either by acetylcholine or bycations present in the solution used to maintain the oocytes. The factthat the block was more pronounced at high agonist concentrationsindicates that at least part of this effect is due to avoltage-dependent channel block by acetylcholine. High concentrations ofacetylcholine and carbamylcholine are known to produce a voltage- andconcentration-dependent channel block of muscle nAChR present in BC₃ H-1cells (Sine and Steinbach, Biophys. J., 46, 277-284, 1984).

Based on its primary structure and electrophysiological properties, thealpha9 protein belongs to the nicotinic family of ligand gated ionchannels which includes subunits for nAChR, GABA_(A), glycine and 5-HT₃receptors. However, as described earlier, in alpha9-injected oocytes,nicotine, muscarine, d-tubocurarine and atropine blockedacetylcholine-evoked current responses. Therefore, the alpha9receptor-channel complex falls into neither the nicotinic nor themuscarinic subdivisions of the pharmacological classification scheme ofcholinergic receptors (P. Taylor in The pharmacalogical basis oftherapeutics, A. Goodman-Gilman, T. H. Rall, A. S. Nies and P. Taylor,eds. (New York:Pergamon Press), pp. 122-149 and 166-186, 1990). Thefinding that both the nicotinic agonist DMPP and the muscarinic agonistOXO-M are capable of eliciting current responses in alpha9-injectedoocytes indicates that the alpha9 receptor exhibits a mixednicotinic-muscarinic pharmacology. In addition, the blockage of alpha9receptors by the glycine receptor antagonist strychnine is unusual. Asimilar effect of strychnine has also been reported on alpha7 and alpha8homomers expressed in Xenopus oocytes (Seguela et al., J. Neurosci., 12,596-604, 1993; Gerzanich et al., Molec. Pharmacol., 45, 212-220, 1994).

The alpha9 protein subunit contains the most conserved amino acidresidues within the proposed acetylcholine binding site of nAChR alphasubunits (Dennis et al., Biochem, 27, 2346-2357, 1988; Galzi et al., J.Biol. Chem., 265, 10430-10437, 1990). Nevertheless, two non-conservativesubstitutions in the alpha9 protein, Phe-99 to Ser and Lys-144 to Thr(position numbers refer to the mature protein, absent the 28 residues ofthe leader sequence), are near the first and second domains of thepostulated agonist binding site for nAChR. These amino acidsubstitutions are likely to be responsible for the distinctpharmacological properties of the alpha9 receptor-channel complex.

To determine the tissue expression pattern of the alpha9 gene, in situhybridization studies were performed. In vitro synthesized RNA derivedfrom coding sequence of the alpha9 genomic clone was hybridized tosagittal sections of rat embryos and coronal sections of adult ratbrains. The presence of transcripts are observed in the hypophysealgland of a rat embryo at stage E16 (see FIGS. 9B and 9D). The alpha9gene expression is observed to be restricted to the pars tuberalis ofthe adenohypophysis, whereas the pars distalis and the neurohypophysisshow no detectable signal. The alpha9 mRNA is also observed to bepresent in the adult rat pars tuberalis, at the ventral surface of themedian eminence (see FIG. 9F). Alpha9 expression is also observedthroughout the E16 rat olfactory mucosa (see FIG. 9B). The alpha9transcripts are detected in the pseudostratified columnar epitheliumthat lines each of the turbinates of the olfactory organ. Additionalexpression is seen in the tongue of the developing rat (FIG. 9B).Finally, in situ hybridization analysis performed on 20 mm coronalsections every 180 mm throughout the adult brain, did not establishalpha9 gene expression in the central nervous system of the rat.

In situ hybridization studies performed on cryostat sections of the ratcochlea indicate that the alpha9 gene is also expressed in the outerhair cell region of all cochlea turns. No expression of the alpha9 genewas observed in spiral ganglion neurons or other supporting structuresof the cochlea (see FIG. 9B).

Previously published neuronal nAChR genes are reported as beingexpressed in the central nervous system of vertebrates (Sargent, Annu.Rev. Neurosci., 16, 403-443, 1993). As disclosed above, in situhybridization studies performed in coronal sections throughout the ratbrain did not establish alpha9 gene expression in the central nervoussystem. Although low levels of alpha9 transcripts or a very restrictedexpression pattern which escaped detection cannot be ruled out, theresults suggest that relative to other nAChR subunits, alpha9 may beinvolved in a distinct subset of cholinergic functions in vivo. In situhybridization studies showed that in the rat the alpha9 subunit gene isexpressed in the pars tuberalis of the pituitary, the olfactoryepithelium, the outer hair cells of the cochlea and the skeletal muscleof the tongue.

The pars tuberalis constitutes an anatomically well defined part of thevertebrate adenohypophysis consisting of peptide-secreting cells,gonadotropes and thyrotropes (Wittkowski et al., Acta Endocrinol., 126,285-290, 1992). Neuroendocrine effects, such as the inhibition ofluteinizing hormone and thyroid-stimulating hormone secretion inresponse to nicotine exposure, has been reported in humans and rats(Fuxe et al., Psychoneuroendocrinol., 14, 19-41, 1989). Although theseeffects have been attributed to the activation of hypothalamic nAChR,the presence of the alpha9 nAChR subunit in the pituitary indicates thatnicotine may exert a direct action on this gland.

It is likely that the olfactory sensory cells receive efferentinnervation that modulates olfactory function (Shirley, Olfaction. Intl.Rev. Neurobiol., 33, 1-53, 1992). A cholinergic modulation has beensuggested, since the application of acetylcholine causes slow electricalpotentials and modifies spike activity in olfactory receptor neurons(Bouvet et al., Neurosci. Res., 5, 214-223, 1988). Although furtherpharmacological characterization of the acetylcholine response inolfactory neurons as well as a more precise localization of the alpha9subunit within the olfactory epithelium are necessary, the presence ofalpha9 transcripts in the olfactory epithelium could provide themolecular basis for the cholinergic effect described.

The alpha9 gene expression in the developing muscle of the tongue isintriguing. With the in situ hybridization studies performed it is notpossible to distinguish whether the signal is actually localized inmuscle fibers or in the surrounding connective tissue. However, alpha9transcripts appear not to be present in all developing skeletal muscles.For example, in situ hybridization studies performed in mid-sagittalsections of rat embryos showed no evidence of alpha9 transcripts in theintercostal or axial muscles.

The overall pharmacological characteristics of the homomeric alpha9receptor expressed in oocytes differ from those of other cloned nAChRs(Boulter, et al., Proc. Natl. Acad. Sci. USA, 84, 7763-7767, 1987;Ballivet, et al., Neuron, 1, 847-852, 1988; Wada, et al., Science, 240,330-334, 1988; Couturier, et al., Neuron, 5, 847-856, 1990; Gerzanich,et al., Molec. Pharmacol., 45, 212-220, 1994).

In order to further investigate the expression pattern of the alpha9gene in the rat cochlea, PCR was performed on cDNA reverse transcribedfrom cochlear total RNA. Two primers specific for the alpha9 sequencewere designed and used to amplify a fragment that spans an intron-exonboundary of the alpha9 gene. As shown on FIG. 8, a fragment of theexpected size (573 bp) was amplified from rat cochlear cDNA with alpha9primers. Restriction endonuclease analysis of the fragment with AccI,HinfI and NcoI, further confirmed that it was derived from alpha9transcripts. Since the alpha9 gene is also transcribed in the ratolfactory epithelium, RNA obtained from this tissue was used as apositive control. Rat sciatic nerve cDNA was included as a negativecontrol to rule out the possibility that, with the parameters used forthe PCR, very low levels of transcripts would be detected in any tissuestudied. Whereas no DNA was amplified from the sciatic nerve usingspecific primers for alpha9 (see FIG. 8), both alpha3 and alpha4subunits could be detected in this tissue with their respective specificprimers.

A possible physiological role for the alpha9 receptor-channel is theefferent cholinergic innervation of cochlea hair cells. Outer hair cellsof the cochlea are implicated in the mechanical amplification of soundin vertebrates (Flock, R. Klinke and R. Hartmann, eds.(Berlin:Springer-Verlag), pp. 2-8, 1983). These cells receive efferentcholinergic innervation. The electrical stimulation of these efferentneurons results in reduction of sensitivity and tuning of the auditorynerve fibers, which in turn could induce protection against acoustictrauma (Brown and Nuttal, J. Physiol. (Lond.), 354, 625-646, 1984;Klinke, Hearing Res., 22, 235-243, 1986; Rajan and Johnstone, BrainRes., 458, 241-255, 1988). The molecular nature of the acetylcholinereceptor involved in the efferent innervation of cochlear hair cells hasnot been described. Although both a non-selective cation channel as wellas a G-protein coupled receptor have been proposed, cholinergic agonistsand antagonists have been of little benefit to characterize thisreceptor as either nicotinic or muscarinic (Housley and Ashmore, Proc.R. Soc. Lond. B, 244, 161-167, 1991; Fuchs and Murrow, Proc. R. Soc.Lond. B, 248, 35-40, 1992; Fuchs and Murrow, J. Neurosci., 12, 800-809,1992; Kakehata et al., J. Physiol. (Lond.), 463, 227-244, 1993;Erostegui et al., Hearing Res., 74, 135-147, 1994). Therefore, whateverthe primary structure for this cholinergic receptor might be, based onits unique pharmacological characteristics it has been suggested that itis of a receptor type not previously described (Fuchs and Murrow, Proc.R. Soc. Lond. B, 248, 35-40, 1992; Erostegui et al., Hearing Res., 74,135-147, 1994).

The results presented herein suggest that the alpha9 receptor is thecholinergic component of the cochlear efferent system. This conclusionis based primarily on the presence of alpha9 transcripts in the haircells of the rat cochlea. Evidence to date suggests that the cochlearefferent system is involved in improving the detection of signal withinbackground noise, protection of the cochlea from noise damage, andattenuating the cochlear response to auditory stimulation when attentionmust be focused elsewhere.

Various experiments have shown that the cholinergic component of thecochlear efferent system may also be involved in aminoglycosideantibiotic ototoxicity. When administered in high doses, theseantibiotics cause outer hair cells (OHC) to degenerate (Govaerts, etal., Toxicology Letters, 52, 227-251, 1990). The results of suchdegeneration ranges from ringing in the ears to total loss of hearing.Current theories regarding the mechanisms whereby aminoglycosides exerttheir ototoxic effect upon the OHCs suggest that the OHCs becomemetabolically destabilized due to a block of intracellular messagingsystems. At the same time, the efferent synapses are also destabilized,and can no longer monitor and control the amount of ACh releasedfollowing stimulation. The end result is that there is anoverstimulation (an excess of ACh) directed toward the destablized OHCs,which results in the degeneration observed (Williams, et al., HearingRes., 30, 11-18, 1987). Thus, ACh, and the alpha9 receptor responsiblefor transducing the efferent signal from the efferent terminal to thehair cell, are intimately involved in releasing the ototoxic potentialof the aminoglycoside antibiotics. Accordingly, antagonists to receptorscomprising at least one alpha9 receptor subunit (i.e., alpha9 blockers)will reduce or eliminate the side-effects of aminoglycoside-inducedototoxicity.

The present invention provides isolated nucleic acids encoding an alpha9nicotinic acetylcholine receptor subunit. The term "nucleic acids" (alsoreferred to as polynucleotides) encompasses RNA as well as single anddouble-stranded DNA and cDNA. As used herein, the phrase "isolatedpolynucleotide" refers to a polynucleotide that has been separated orremoved from its natural environment. One means of isolating apolynucleotide encoding an alpha9 nAChR receptor subunit is to probe amammalian genomic library with a DNA probe using methods well known inthe art. DNA probes derived from the alpha9 receptor gene particularlyuseful for this purpose. DNA and cDNA molecules that encode alpha9receptors can be used to obtain complementary genomic DNA, cDNA or RNAfrom human, mammalian, or other animal sources. Such molecules can alsobe used to isolate related cDNA or genomic clones by screening cDNA orgenomic libraries, by methods described in more detail below. Inventionnucleic acids include nucleotide sequences that are substantially thesame as the nucleotide sequence shown in FIG. 1 (see also SEQ ID NO:1).The present invention also encompasses nucleic acids which aredegenerate variants of the nucleotide sequence shown in FIG. 1 (and SEQID NO:1).

The term "degenerate variants" refers to nucleic acids encoding alpha9nAChR subunits that, by virtue of the degeneracy of the genetic code, donot necessarily hybridize to the invention nucleic acids under specifiedhybridization conditions. Preferred nucleic acids encoding inventionpolypeptide(s) or proteins(s) are comprised of nucleotides that encodesubstantially the same amino acid sequence set forth in FIG. 1 (see alsoSEQ ID NO:2). Alternatively, preferred nucleic acids encoding inventionpolypeptide(s) hybridize under high stringency conditions tosubstantially the entire sequence, or substantial portions (i.e.,typically at least 25-30 contiguous nucleotides) of the nucleotidesequence set forth in FIG. 1 (see also SEQ ID NO:1).

Stringency of hybridization, as used herein, refers to conditions underwhich polynucleotide hybrids are stable. As known to those of skill inthe art, the stability of hybrids is a function of sodium ionconcentration and temperature. (See, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual 2d Ed. (Cold Spring HarborLaboratory, 1989; incorporated herein by reference).

The invention provides isolated alpha9 nicotinic acetylcholine receptorsubunit peptide, polypeptide and/or protein encoded by invention nucleicacids and alpha9 nicotinic acetylcholine receptor comprising saidsubunit. The alpha9 nAChR subunit comprises a protein of approximately451 amino acids in length. The amino acid sequence of the alpha9 subunitis set forth in FIG. 1 (and in SEQ ID NO:2).

As used herein, the term "isolated protein" refers to a protein free ofcellular components and/or contaminants normally associated with aprotein in its native in vivo environment. Invention polypeptides and/orproteins include naturally occurring allelic variants, as well asrecombinant forms thereof. The alpha9 nAChR polypeptide can be isolatedusing various methods well known to those of skill in the art. Themethods available for the isolation and purification of inventionproteins include, precipitation, gel filtration, ion-exchange,reverse-phase and affinity chromatography. Other well-known methods aredescribed in Deutscher et al., Guide to Protein Purification: Methods inEnzymology Vol. 182, (Academic Press, 1990), which is incorporatedherein by reference. Alternatively, isolated polypeptides of the presentinvention can be obtained using well-known recombinant methods asdescribed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual 2d Ed. (Cold Spring Harbor Laboratory, 1989;incorporated herein by reference).

Invention polypeptide(s) can be produced by expressing nucleic acidsencoding the alpha9 nAChR subunit in a suitable host cell, such as abacterial cell, a yeast cell, an amphibian cell (i.e., oocyte), or amammalian cell, using methods well known in the art. The expressedpolypeptide can be recovered using well-known methods. Inventionpolypeptides can be isolated directly from cells that have beentransformed with expression vectors, described below in more detail. Theinvention polypeptide, biologically active fragments, and functionalequivalents thereof can also be produced by chemical synthesis. As usedherein, "biologically active fragment" refers to any portion of thealpha9 polypeptide represented by the amino acid sequence in FIG. 1 (seealso SEQ ID NO:2) that can assemble into a cationic channel activated byacetylcholine and permeable to calcium. Synthetic polypeptides can beproduced, for example, using Applied Biosystems, Inc. Model 430A or 431Aautomatic peptide synthesizer (Foster City, Calif.) employing thechemistry provided by the manufacturer.

As used herein, the phrase "nicotinic acetylcholine receptor (nAChR)subunit" refers to recombinantly expressed/produced (i.e., isolated orsubstantially pure) protein that contains four highly hydrophobicregions which predict membrane spanning regions and cysteine residues atpositions 127, 141, 191 and 192 (referring to the mature peptide, notincluding the 28 amino acid leader sequence). Such protein subunitsassemble into a cationic channel which is activated by acetylcholine.Invention nAChR subunits include variants thereof encoded by mRNAgenerated by alternative splicing of a primary transcript, as well asbiologically active fragments.

The alpha9 nAChR subunit of the invention contributes to the formationof a functional receptor, as assessed by methods described herein, bycombining with at least one additional nAChR subunit of the same ordifferent type. As used herein, the phrase "functional receptor" meansthat the binding of a ligand, for example, acetylcholine (ACh), causesthe receptor ion channel to open thereby permitting cations, such asCa²⁺, as well as Na⁺ and K⁺, to enter the cell. Agonist activation of a"functional invention receptor" induces the receptor.

Modification of invention nucleic acids, polypeptides or proteins withthe following phrases: "recombinantly expressed/produced", "isolated",or "substantially pure", encompasses nucleic acids, peptides,polypeptides or proteins that have been produced in such form by thehand of man, and are thus separated from their native in vivo cellularenvironment. As a result of this human intervention, the recombinantnucleic acids, polypeptides and proteins of the invention are useful inways that the corresponding naturally occurring molecules are not, suchas identification of compounds as potential drugs.

Sequences having "substantial sequence homology" are intended to referto nucleotide sequences that share at least about 90% identity withinvention nucleic acids; and amino acid sequences that typically shareat least about 95% amino acid identity with invention polypeptides. Itis recognized, however, that polypeptides or nucleic acids containingless than the above-described levels of homology arising as splicevariants or generated by conservative amino acid substitutions, or bysubstitution of degenerate codons are also encompassed within the scopeof the present invention.

The present invention also provides nucleic acids encoding alpha9receptor subunit(s) operatively linked to a promoter, as well as otherregulatory sequences. As used herein, the phrase "operatively linked"refers to the functional relationship of the nucleic acid withregulatory and effector sequences, such as promoters, enhancers,transcriptional and translational stop sites, and other signalsequences. Specifically operative linkage of a nucleic acid to apromoter refers to the physical and functional relationship between thenucleic acid and the promoter such that transcription of DNA isinitiated from the promoter by an RNA polymerase that specificallyrecognizes and binds to the promoter.

Suitable promoters include specific sequences that are sufficient forRNA polymerase recognition, binding and transcription initiation.Additionally, suitable promoters include sequences that modulate therecognition, binding and transcription initiation activity of RNApolymerase. Such sequences may be cis acting or may be responsive totrans acting factors. Depending upon the nature of the regulation,promoters may be constitutive or regulated. Examples of promoters areSP6, T4, T7, SV40 early promoter, cytomegalovirus (CMV) promoter, mousemammary tumor virus (MMTV) steroid-inducible promoter, Moloney murineleukemia virus (MMLV) promoter, and the like.

Vectors employed in the present invention contain both a promoter and acloning site into which nucleic acid encoding alpha9 receptor subunit(s)can be operatively linked. Such vectors, which are well known in theart, are capable of transcribing RNA in vitro or in vivo, and arecommercially available from sources such as Stratagene (La Jolla,Calif.) and Promega Biotech (Madison, Wis.). In order to optimizeexpression and/or in vitro transcription, it may be necessary to remove,add or alter 5' and/or 3' untranslated portions of the clones toeliminate extra, potentially inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5' of the start codon to enhance expression. (See, forexample, Kozak, J. Biol. Chem. 266:19867 (1991)). Similarly, alternativecodons, encoding the same amino acid, can be substituted for nativecodons of the alpha9 nAChR subunit in order to enhance transcription(e.g., the codon preference of the host cell can be adopted, thepresence of G-C rich domains can be reduced, and the like).

Examples of suitable vectors that may be employed in the presentinvention include viruses, such as baculoviruses and retroviruses,bacteriophages, cosmids, plasmids and other recombination vehiclestypically used in the art. Invention nucleic acids are inserted intovector genomes using methods well known in the art. For example, insertand vector DNA can be contacted, under suitable conditions, with arestriction enzyme to create complementary ends on each molecule thatcan pair with each other and be joined together with a ligase.Alternatively, synthetic linkers can be ligated to the termini ofrestricted invention nucleic acids. These synthetic linkers containnucleic acid sequences that correspond to a particular restriction sitein the vector DNA. Additionally, a nucleic acid containing a terminationcodon and an appropriate restriction site can be ligated into a vectorcontaining, for example, some or all of the following: a selectablemarker gene, such as the neomycin gene for selection of stable ortransient transfectants in mammalian cells; enhancer/promoter sequencesfrom the immediate early gene of human CMV for high levels oftranscription; transcription termination and RNA processing signals fromSV40 for mRNA stability; SV40 polyoma origins of replication and ColE1for proper episomal replication; versatile multiple cloning sites; andT7 and SP6 RNA promoters for in vitro transcription of sense andantisense RNA. Other means are well known and available in the art.

Also provided are vectors comprising nucleic acid encoding alpha9 nAChRsubunit, which vectors are adapted for expression in a bacterial cell, ayeast cell, an amphibian cell (i.e., oocyte), a mammalian cell or otheranimal cells. Such vectors additionally comprise regulatory elementsnecessary for expression of nucleic acid in the bacterial, yeast,amphibian, mammalian or animal cells located relative to the nucleicacid encoding alpha9 nAChR subunit so as to permit expression thereof.As used herein, "expression" refers to the process by which nucleicacids are transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the nucleic acid is derived from genomicDNA, expression may include splicing of the mRNA, if an appropriateeucaryotic host is selected. Regulatory elements required for expressioninclude promoter sequences to bind RNA polymerase and transcriptioninitiation sequences for ribosome binding. For example, a bacterialexpression vector might include a promoter such as the lac promoter, theShine-Dalgarno transcription initiation sequence and the start codon AUG(Sambrook et al., Molecular Cloning: A Laboratory Manual 2d Ed. (ColdSpring Harbor Laboratory, 1989; incorporated herein by reference).Similarly, a eucaryotic expression vector might include a heterologousor homologous promoter for RNA polymerase II, a downstreampolyadenylation signal, the start codon AUG, and a termination codon fordetachment of the ribosome. Such vectors can be obtained commercially orassembled from available sequences and by methods well known in the art.

This invention also provides a transformed host that expressesrecombinant alpha9 nicotinic acetylcholine receptor. Such a host hasbeen transformed with a nucleic acid encoding alpha9 nAChR subunit. Anexample of a transformed host according to the present invention is amammalian cell comprising a plasmid specifically adapted for expressionin such a cell. The plasmid contains a nucleic acid encoding an alpha9nAChR subunit and the regulatory elements necessary for expression ofthe subunit. Suitable mammalian cells that may be utilized in thepresent invention include, for example, mouse fibroblast NIH3T3 cells,CHO cells, HeLa cells, Ltk⁻ cells, PC12 and N2A neuronal cells, HEK-293kidney cells and CG4 glial cells. Host cells may be transformed withplasmids such as those described supra by methods well known in the artsuch as calcium phosphate precipitation, DEAE-dextran, electroporation,microinjection or lipofection. Other suitable hosts that may be employedin the present invention are oocytes, particularly Xenopus oocytes.

Nicotinic acetylcholine receptors, according to the invention, arerecombinantly expressed in a host cell containing at least one alpha9subunit. Recombinant receptors may be homomeric or heteromeric. Thus, atransformed host cell can recombinantly express a receptor containingonly alpha9 subunits, or containing at least one alpha9 subunit and oneor more other nAChR subunits.

The present invention also provides nucleic acid probes. Such probescomprise a polynucleotide capable of specifically hybridizing with asequence encoding an alpha9 nAChR subunit. As used herein, the term"probe" refers to single-stranded or double-stranded DNA or RNA that hasa sequence of nucleotides that includes at least 14 contiguous bases setforth in FIG. 1 (see also SEQ ID NO:1). Probes used to distinguish thealpha9 subunit from other alpha nAChRs subunits will preferably consistof at least 14 contiguous bases from the cytoplasmic loop region of thealpha9 nucleotide sequence. Alternatively, probes that are to be used tofind additional subunits of the nAChR family will preferably consist ofat least 14 contiguous bases from a membrane spanning region of thealpha9 nucleotide sequence.

As used herein, the phrase "specifically hybridizing" encompasses theability of a polynucleotide to recognize a nucleic acid sequence that iscomplementary thereto and to form double-helical segments via hydrogenbonding between complementary base pairs. Nucleic acid probe technologyis well known to those skilled in the art who will readily appreciatethat such probes may vary greatly in length and may be labeled with adetectable agent, such as a radioisotope, a fluorescent dye, and thelike, to facilitate detection of the probe. Invention probes are usefulto detect the presence of nucleic acids encoding the alpha9 nAChRsubunit. For example, the probes can be used for in situ hybridizationsto identify specific tissues in which the alpha9 nAChR subunit gene isexpressed. Additionally, oligonucleotides complementary to nucleic acidsencoding the alpha9 nAChR subunit are useful for detecting the alpha9gene and associated mRNA, or for the isolation of related genes usinghomology screening of genomic or cDNA libraries, or by usingamplification techniques well known to those of skill in the art.

The invention further provides antisense oligonucleotides having asequence capable of binding specifically with any portion of an mRNAthat encodes the alpha9 nAChR subunit so as to prevent translation ofthe mRNA. Antisense oligonucleotides may also contain a sequence capableof binding specifically with any portion of the cDNA encoding the alpha9subunit. As used herein, the phrase "binding specifically" encompassesthe ability of a nucleic acid sequence to recognize a complementarynucleic acid sequence and to form double-helical segments therewith viathe formation of hydrogen bonds between the complementary base pairs.

Also provided by the present invention are compositions comprising anamount of an invention antisense oligonucleotide effective to reduceexpression of the alpha9 nAChR subunit wherein said antisenseoligonucleotide is capable of binding with mRNA encoding the alpha9nAChR receptor so as to prevent its translation. Compositions providedby the present invention comprise an acceptable hydrophobic carriercapable of passing through cell membranes and may also comprise astructure which binds to a receptor specific for a selected cell typeand is thereby taken up by cells of the selected cell type. Thestructure may be part of a protein known to bind to a cell-type specificreceptor.

Antisense oligonucleotide compositions (AOCs) according to the presentinvention are designed to be stable in the blood stream foradministration to a subject by injection, or in laboratory cell cultureconditions. The physical and chemical properties of the AOC are selectedso that the composition is capable of passing through the cell membranein order to enter the cell cytoplasm. Such a composition can be designedto include small, hydrophobic chemical structures, or alternatively,specific cell transport systems which facilitate and transport the AOCinto the cell. In addition, the AOC can be designed for administrationonly to certain selected cell populations by targeting the AOC to bebound and taken up by select cell populations. Targeting can beaccomplished by designing cell specific AOCs to bind to a receptor foundonly in a certain cell type, as discussed supra. Alternatively, an AOCcan also be designed to recognize and selectively bind to a target mRNAsequence. In the latter instance, targeting is accomplished, forexample, by employing a sequence contained within the sequence shown inFIG. 1 (SEQ ID NO:1). The AOC is designed to inactivate the target mRNAsequence by (1) binding to target mRNA and inducing degradation of themRNA by, for example, RNase I digestion, or (2) inhibiting translationof target mRNA by interfering with the binding of translation-regulatingfactors or ribosomes, or by inclusion of other chemical structures, suchas ribozyme sequences or reactive chemical groups which either degradeor chemically modify the target mRNA. AOCs have been shown to be capableof such properties when directed against mRNA targets (see Cohen et al.,TIPS, 10:435 (1989) and Weintraub, Sci. American, January (1990), pp.40; both incorporated herein by reference).

The invention also provides antibodies having specific reactivity withalpha9 nAChR polypeptides and/or proteins of the subject invention.Active fragments of antibodies are encompassed within the definition of"antibody".

The antibodies of the invention can be produced by methods known in theart. For example, polyclonal and monoclonal antibodies can be producedby methods described, for example, in Harlow and Lane, Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory 1988), which isincorporated herein by reference. The alpha9 protein of the invention,or portions thereof, can be used as the immunogen in generating suchantibodies. Alternatively, synthetic peptides can be prepared (usingcommercially available synthesizers) and used as immunogens. Amino acidsequences can be analyzed by methods well known in the art to determinewhether they encode hydrophobic or hydrophilic domains of thecorresponding alpha9 invention protein. Altered antibodies such aschimeric, humanized, CDR-grafted or bifunctional antibodies can also beproduced by methods well known in the art. Such antibodies can also beproduced by hybridoma, chemical synthesis or recombinant methodsdescribed, for example, in (Sambrook et al., Molecular Cloning: ALaboratory Manual 2d Ed. (Cold Spring Harbor Laboratory, 1989);incorporated herein by reference and Harlow and Lane, supra). Bothanti-peptide and anti-fusion protein antibodies can be used. (see, forexample, Bahouth et al., Trends Pharmacol. Sci. 12:338 (1991); Ausubelet al., Current Protocols in Molecular Biology (John Wiley and Sons, NY1989) which are incorporated herein by reference).

The invention antibodies have various uses, such as, for example,isolation of the alpha9 invention receptor. Additionally, the antibodiesare useful for detecting the presence of the alpha9 receptor, as well asanalysis of receptor localization, subunit composition, and structure offunctional domains. A method for detecting the presence of alpha9 nAChRson the surface of a cell comprises contacting the cell with an antibodythat specifically binds alpha9 nACh receptor and detecting the presenceof the bound antibody on the cell surface. With respect to the detectionof alpha9 receptors, the invention antibodies can be used, for example,for in vitro diagnostic or in vivo imaging methods.

Immunological procedures useful for in vitro detection of alpha9receptor in a sample include immunoassays that employ a detectableantibody. Such immunoassays include, for example, ELISA, Pandexmicrofluorimetric assay, agglutination assays, flow cytometry, serumdiagnostic assays and immunohistochemical staining procedures which arewell known in the art. An antibody can be made detectable by variousmeans well known in the art. For example, a detectable marker can bedirectly or indirectly attached to the antibody. Useful markers include,for example, radionuclides, enzymes, fluorogens, chromogens andchemiluminescent labels.

Further, invention antibodies can be used to modulate the ion channelactivity of the alpha9 receptor in animals and humans as well asbiological tissues isolated therefrom. Accordingly, the inventionprovides compositions comprising a carrier and an amount of an antibodyhaving specificity for the alpha9 receptor effective to block binding ofnaturally occurring ligands to the receptor. A monoclonal antibodydirected to an epitope of an alpha9 receptor present on the surface of acell wherein said antibody has an amino acid sequence substantially thesame as an amino acid sequence set forth in Sequence ID No. 2 can beuseful for this purpose.

The invention further provides a transgenic non-human mammal capable ofexpressing nucleic acid encoding alpha9 protein. Also provided aretransgenic non-human mammals incapable of expressing nucleic acidencoding biologically functional alpha9 protein or alternatively,capable only of expressing alpha9 protein that is biologically deficientin some respect. Varying degrees of disfunctionality are achievedthrough manipulation of alpha9 nucleic acid to encode a mutated protein.

The present invention also provides a transgenic non-human mammal havinga genome comprising antisense nucleic acid which is transcribed intoantisense mRNA complementary to alpha9 mRNA. Such antisense mRNAhybridizes to alpha9 mRNA and reduces the translation thereof.

Nucleic acids employed in transgenic animals of the invention may beassociated with an inducible promoter and/or tissue specific regulatoryelements, so that expression can be induced, or restricted to specificcell types. Examples of suitable promoters are the metallothioneinpromoter and the L7 promoter.

The transfer of nucleic acid material into mammalian hosts for thepurpose of generating transgenic animals can be accomplished bymicroinjection, retroviral infection or other means well known to thoseskilled in the art, of the material into appropriate fertilized embryos.(See, for example, Hogan et al., Manipulating the Mouse Embryo: ALaboratory Manual (Cold Spring Harbor Laboratory, 1986). Homologousrecombination can also be used for the generation of transgenic animalsaccording to the present invention. Homologous recombination techniquesare well known in the art. Homologous recombination replaces a native(endogenous) gene with a recombinant or mutated gene to produce ananimal that cannot express a native (endogenous) alpha9 receptor subunitbut can express, for example, a mutated receptor subunit. In contrast tohomologous recombination, microinjection adds genes to the host genome,without removing host genes. Microinjection can produce a transgenicanimal that is capable of expressing both endogenous and exogenousalpha9 receptor subunits. Transgenic animal model systems are useful forin vivo screening of compounds for identification of receptor-specificligands, i.e., agonists and antagonists, which activate or inhibitreceptor responses.

Nucleic acids, oligonucleotides (including antisense), vectorscontaining same, transformed hosts, receptor subunits and combinationsthereof, as well as antibodies of the present invention, can be used toscreen compounds in vitro to identify those compounds which function asagonists or antagonists of alpha9 receptor subunits of the invention.Such in vitro screening assays provide useful information regarding thefunction and activity of alpha9 receptor subunits of the invention,which can facilitate the identification and design of drugs that arecapable of specific interaction with one or more types of receptorsubunits or receptor subtypes.

The present invention also provides a method for identifying compoundswhich bind to alpha9 nicotinic acetylcholine receptor subunits. In sucha method, invention receptor subunits may be employed in a competitivebinding assay. Such an assay can accommodate the rapid screening of alarge number of compounds to determine which compounds, if any, arecapable of binding to the alpha9 nAChR subunit. Subsequently, moredetailed assays can be carried out with those compounds found to bind,to further determine whether such compounds act as agonists orantagonists of invention receptors (i.e., nAChRs comprising at least onealpha9 subunit).

The present invention still further provides a bioassay for identifyingcompounds which modulate the activity of receptors of the invention(i.e., nAChRs comprising at least one alpha9 subunit). In oneembodiment, the bioassay is conducted by providing cells expressingreceptor comprising at least one alpha9 subunit with at least onepotential agonist and thereafter monitoring the cells for changes in ionchannel activity. In yet another embodiment, the bioassay is conductedby contacting cells expressing receptor comprising at least one alpha9subunit with a constant amount of a known alpha9 agonist and increasingamounts of at least one potential antagonist and thereafter monitoringthe cells for changes in ion channel activity.

The present invention also provides a bioassay for identifying compoundswhich modulate the regulatory regions of the alpha9 nAChR subunit gene.Such an assay is conducted utilizing mammalian cells transformed with anucleic acid construct comprising at least a portion of the regulatoryregion of the alpha9 gene operatively associated with a reporter gene.The transformed cells are contacted with at least one compound whereinthe ability of said compound to modulate the regulatory region isunknown. Thereafter, the cells are monitored for expression of thereporter gene. Suitable reporter genes that may be employed include, forexample, the chloramphenicol acetyltransferase gene, the luciferasegene, and the like.

A compound or a signal that "modulates the activity" of an inventionreceptor refers to a compound or a signal that alters the activity ofthe alpha9 receptor so that the receptor is different in the presence ofthe compound or signal than in the absence of the compound or signal.Compounds affecting modulation include agonists and antagonists. Anagonist encompasses a compound such as acetylcholine, that activatesalpha9 receptor function. Alternatively, an antagonist includes acompound that interferes with alpha9 receptor function. Typically, theeffect of an antagonist is observed as a blocking of agonist-inducedreceptor activation. Antagonists include competitive as well asnon-competitive antagonists. A competitive antagonist (or competitiveblocker) interacts with or near the site specific for agonist binding. Anon-competitive antagonist or blocker inactivates the function of thereceptor by interacting with a site other than the agonist interactionsite.

As understood by those of skill in the art, bioassay methods foridentifying compounds that modulate nAChR activity generally requirecomparison to a control. One type of "control" is a cell or culture thatis treated substantially the same as the test cell or test cultureexposed to the compound, with the distinction that the "control" cell orculture is not exposed to the compound. For example, in methods that usevoltage clamp electrophysiological procedures, the same cell can betested in the presence or absence of compound, by merely changing theexternal solution bathing the cell. Another type of "control" cell orculture that can be employed is a cell or culture that is identical totransfected cells, with the exception that the "control" cell or culturedoes not express functional alpha9 nACh receptor subunit. Accordingly,the response of the transfected cell to compound is compared to theresponse (or lack thereof) of the "control" cell or culture to the samecompound under the same reaction conditions.

In still another embodiment of the present invention, the ion channelactivity of alpha9 nAChR can be modulated by contacting the receptorswith an effective amount of at least one compound identified by any ofthe above-described bioassays.

The following examples are intended to illustrate, but not limit, thepresent invention.

EXAMPLE I Screening of Genomic Libraries

A full-length alpha7 nAChR subunit cDNA (Seguela, et al., J. Neurosci.,13, 596-604, 1993) was used to screen 5×10⁵ clones of a lambdaCharon 4Arat genomic library (obtained from Dr. James Eberwine, Department ofPharmacology, University of Pennsylvania Medical School, Philadelphia,Pa.). Hybridization was carried out at 65° C. in 1M NaCl, 50 mMTris-HCl, pH 8.0, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA and0.1% (w/v) each of Ficoll, polyvinylpyrrolidone and bovine serumalbumin. Filters were washed at 45° C. in 2×SSPE (1×SSPE is 180 mM NaCl,9 mM Na₂ HPO₄, 0.9 mM NaH₂ PO₄ and 1 mM EDTA, pH 8.0). A clone of ˜16 kbcontaining exons IV and V of the alpha9 subunit gene was isolated.

EXAMPLE II Screening of a cDNA Library

A PCR fragment derived from coding sequences (nucleotides 283 to 806,FIG. 1; i.e., nucleotides 455 to 979 of SEQ ID NO:1) of the rat genomicclone described in EXAMPLE I was used as a probe to screen 1×10⁶ plaquesof a lambdaNM1149 adult rat olfactory epithelium cDNA library (obtainedfrom Dr. Heinz Breer, and Dr. Klaus Raming, UniversityStuttgart-Hohenheim, Institute of Zoophysiology, Stuttgart, Germany).Hybridization was as described in EXAMPLE I and filters were washed at65° C. in 0.2×SSPE. Four independent clones were isolated, one containeda full-length alpha9 cDNA (FIG. 1). The alpha9 cDNA consists of an 87 bp5' untranslated region, an open reading frame of 1437 bp and 413 bp of3' untranslated region. The full length alpha9 cDNA was used as a probeto screen two mouse (129SvJ) genomic libraries constructed in phagevectors lambda DASHII and lambda FIXII. Two overlapping genomic cloneswere obtained (FIG. 2). These clones, spanning the entire codingsequence of the alpha9 subunit gene were cloned into plasmid vectors andthe alpha9 subunit gene structure was determined by sequencing acrossthe intron-exon boundaries.

EXAMPLE III Nucleotide Sequence Determination and Analysis

The alpha9 subunit cDNA clone was sequenced using the Sequenase 2.0 kit(United States Biochemical, Cleveland, Ohio) and syntheticoligonucleotide primers. A comparison of the alpha9 amino acid sequenceswith other nAChR alpha subunits was made using sequence analysissoftware from the University of Wisconsin Genetics Computer GroupDevereux, et al., Nucl. Acids. Res., 12, 387-395, 1984!. The percentsequence identity between paired sequences was calculated by dividingthe number of identical residues by the total number of residues in theshorter of the sequences and multiplying the quotient by 100.

EXAMPLE IV Electrophysiological Procedures

A full-length alpha9 cDNA suitable for Xenopus oocyte expression studieswas constructed by subcloning the fragment from nucleotide -94 to 1766(FIG. 1; i.e., residues 79 to 1938 as presented in SEQ ID NO:1) into theexpression vector pGEMHE (Liman, et al., Neuron, 9, 861-871, 1992). cRNAwas synthesized using the mMessage mMachine transcription kit (Ambion,Austin, Tx.), with plasmid linearized with NheI.

The isolation and maintenance of oocytes has been previously described(Boulter, et al., Proc. Natl. Acad. Sci. USA, 84, 7763-7767, 1987). Eachoocyte was injected with 1 to 10 ng of cRNA. Electrophysiologicalrecordings were performed 2 to 7 days after injection, undertwo-electrode voltage clamp with an Axoclamp 2A amplifier (AxonInstruments, Foster City, Calif.). Voltage electrodes were filled with3M KCl and had a resistance of ˜10 MΩ; current electrodes were filledwith 0.3M KCl and had a resistance of ˜1 MΩ. Unless otherwise stated,the holding potential was -50 mV. I-V relationships were obtained withpClamp 5.5 software (Axon Instruments), by applying 2 second voltageramps in the presence of agonist and subtracting the control averagevalues obtained before and after agonist application. All records weredigitized and stored on a computer. Data was analyzed using softwarethat was designed and provided by Dr. S. Traynelis (The Salk Institutefor Biological Studies, La Jolla, Calif.).

Oocytes were continuously superfused in frog Ringer's solution (10 mMHEPES, pH 7.2, 115 mM NaCl, 1.8 mM CaCl₂ and 2.5 mM KCl). No responseswere observed by the application of drugs to uninjected oocytes. For theinhibition curves (see FIGS. 4B, 5A and 5B), antagonists were coappliedwith 10 μM acetylcholine. In the case of α-bungarotoxin andκ-bungarotoxin (see FIGS. 6A and 6B), oocytes were preincubated withthese drugs for 30 minutes. The mean and standard error of the mean ofpeak current responses of at least four oocytes per experiment arerepresented in the Figures. All curve fits were done using Sigma Plotsoftware (Jandel Scientific) with the following equations:

    (i) Response (for concentration-response curves)= (max-min)/(1+(EC.sub.50 /concentration).sup.n)!+min,

and

    (ii) Response (for concentration-inhibition curves)= (max-min)/(1+(concentration/IC.sub.50).sup.n)!+min.

Atropine sulfate, (-)-nicotine ditartrate, (+)-muscarine chloride,strychnine hydrochloride and oxotremorine-M were obtained from RBI(Natick, Mass.), kappa-Bungarotoxin was donated by Dr. V. Chiappinelli(St. Louis University Medical Center, St. Louis, Mo.). All other drugswere obtained from Sigma Chemical Co. (St. Louis, Mo.). Drugs weredissolved in frog Ringer's solution. Bovine serum albumin (100 mg/ml)was added to the toxin solutions.

EXAMPLE V In situ Hybridization

Experiments were carried out using mid-sagittal sections of rat E16embryos (Hybrid-ready tissue, Novagen, Madison, Wis.) and 20 μm thickcoronal sections of adult rat brains, according to the protocoldescribed by Simmons, et al. in J. Histotechnol., 12, 169-181, 1989.Either ³⁵ S- or ³² P-labeled RNA probes were derived from the alpha9cDNA (e.g., nucleotides 283 to 806, FIG. 1; i.e., nucleotides 455 to 979of SEQ ID NO:1). Hybridization was carried out at 65° C. and finalwashes were carried out at 72° C. in 0.1×SSC (1×SSC is 180 mM NaCl and17 mM sodium citrate, pH 7.0). Slides were dipped in Kodak NTB-2emulsion, developed in Kodak D19 after 3 weeks exposure at 4° C. andsubsequently Nissl stained.

EXAMPLE VI Amplification Reactions

Tissues were obtained from adult Sprague Dawley rats. The animals weredecapitated and the tissues were quickly dissected and immersed inliquid nitrogen. Total RNA was isolated according to Chomczynski andSacchi (see Analytical Biochem., 162, 156-159, 1987), using the TRIzolreagent (Gibco BRL, Gaithersburg, Md.). First strand cDNA wassynthesized from 2 μg of total RNA with the Superscript PreamplificationSystem (Gibco BRL). An aliquot containing 50 ng of cDNA was used astemplate in amplification reactions. The following specific primers foralpha9 were employed: sense primer, nucleotides 778 to 802; antisenseprimer, nucleotide 1353 to 1327 (FIG. 1; nucleotides 951 to 975 andnucleotides 1526 to 1500, respectively, of SEQ ID NO:1). The predictedfragment spans one intron-exon boundary. A 573 base pair band isexpected in the case of amplification from cDNA, whereas a fragment of˜1450 bp would result from the amplification of contaminant genomic DNA.Reactions were done in the following reaction mixture: 5 U of Tag DNApolymerase, 5 U of Tag enhancer (Stratagene, La Jolla, Calif.), 5 μM ofeach primer, 50 μM each of dATP, dGTP, dCTP and dTTP, 20 mM Tris-HCl, pH8.5, 10 mM (H₄ N)₂ SO₄, 2 mM MgSO₄, 0.1% Triton X-100 and 0.1 mg/mlbovine serum albumin. Cycle parameters were: 2 min. at 95° C. followedby 34 cycles each of 1 min. at 55° C., 1 min. at 72° C., 30 sec. at 95°C. and a final cycle of 1 min. at 55° C., 5 min. at 72° C.

EXAMPLE VII Detection of alpha9 Transcripts in Rat Cochlea

To determine if the alpha9 gene is expressed in rat cochlea,amplification reactions were performed on cDNA reverse transcribed fromcochlear total RNA. As described in Example V, two primers specific forthe alpha9 sequence were employed in order to amplify a fragment thatspans an intron-exon boundary and additionally to avoid possibleamplification from genomic DNA. Since alpha9 is present in the ratolfactory epithelium, cDNA obtained from this tissue was used as apositive control. Sciatic nerve cDNA was included to rule out thepossibility that, with the parameters used for the amplificationreactions, very small amounts of transcripts would be detected in anytissue studied. Whereas no DNA was amplified from the sciatic nerveusing specific primers for alpha9 (FIG. 8), both alpha3 and alpha4subunits could be detected in this tissue with the respective specificprimers.

A fragment of the expected size (573 bp) for amplification from alpha9cDNA was obtained in the rat cochlea. Restriction endonuclease analysisof the fragment with AccI, HinfI and NcoI, further confirmed that thisfragment had been derived from alpha9 transcripts.

Although the invention has been described with reference to the specificembodiments, those skilled in the art will readily appreciate that thespecific experiments taught hereinabove are only illustrative of theinvention. It should be understood that various modifications andvariations can be made without departing from the spirit and scope ofthe invention.

SUMMARY OF SEQUENCES

Sequence ID No. 1 is the nucleic acid sequence (and the deduced aminoacid sequence) of a cDNA encoding alpha9 nicotinic acetylcholinereceptor subunit of the present invention.

Sequence ID No. 2 is the deduced amino acid sequence of the alpha9nicotinic acetylcholine receptor subunit of the present invention.

Sequence ID No. 3 is the amino acid sequence of the rat alpha1 nicotinicacetylcholine receptor subunit.

Sequence ID No. 4 is the amino acid sequence of the rat alpha2 nicotinicacetylcholine receptor subunit.

Sequence ID No. 5 is the amino acid sequence of the rat alpha3 nicotinicacetylcholine receptor subunit.

Sequence ID No. 6 is the amino acid sequence of the rat alpha4 nicotinicacetylcholine receptor subunit.

Sequence ID No. 7 is the amino acid sequence of the rat alpha7 nicotinicacetylcholine receptor subunit.

Sequence ID No. 8 is the amino acid sequence of the chick alpha8nicotinic acetylcholine receptor subunit.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1938 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ALPHA 9                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 89..1525                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CGGTGGCAGTGAGGGTGTTTTGAGCCCTTCACAGACAGAAGTGGGAGTCCTCGCTGTCTG60                CCTGACACATTCTACATGTTGGGAAAAGATGAACCGGCCCCATTCCTGCCTC112                       MetAsnArgProHisSerCysLeu                                                      15                                                                            TCCTTTTGCTGGATGTATTTTGCTGCTTCTGGAATCAGAGCCGTAGAG160                           SerPheCysTrpMetTyrPheAlaAlaSerGlyIleArgAlaValGlu                              101520                                                                        ACAGCAAATGGGAAATATGCTCAGAAATTGTTCAGCGATCTTTTTGAA208                           ThrAlaAsnGlyLysTyrAlaGlnLysLeuPheSerAspLeuPheGlu                              25303540                                                                      GACTACTCCAGTGCTCTGCGTCCAGTCGAGGATACGGACGCGGTGCTG256                           AspTyrSerSerAlaLeuArgProValGluAspThrAspAlaValLeu                              455055                                                                        AATGTTACACTGCAGGTCACGCTCTCCCAGATAAAGGACATGGACGAG304                           AsnValThrLeuGlnValThrLeuSerGlnIleLysAspMetAspGlu                              606570                                                                        AGAAACCAGATTCTGACAGCCTATCTATGGATCCGCCAAACCTGGCAC352                           ArgAsnGlnIleLeuThrAlaTyrLeuTrpIleArgGlnThrTrpHis                              758085                                                                        GATGCGTACCTCACGTGGGATCGAGACCAGTATGATAGGCTGGACTCC400                           AspAlaTyrLeuThrTrpAspArgAspGlnTyrAspArgLeuAspSer                              9095100                                                                       ATCAGGATTCCCAGCGATCTGGTGTGGAGGCCGGACATTGTCCTATAC448                           IleArgIleProSerAspLeuValTrpArgProAspIleValLeuTyr                              105110115120                                                                  AACAAGGCTGACGATGAGTCTTCAGAGCCTGTGAACACCAATGTGGTG496                           AsnLysAlaAspAspGluSerSerGluProValAsnThrAsnValVal                              125130135                                                                     CTGCGATATGATGGGCTCATCACCTGGGACTCACCGGCCATCACCAAA544                           LeuArgTyrAspGlyLeuIleThrTrpAspSerProAlaIleThrLys                              140145150                                                                     AGCTCCTGTGTGGTGGATGTCACCTACTTCCCTTTTGACAGCCAGCAG592                           SerSerCysValValAspValThrTyrPheProPheAspSerGlnGln                              155160165                                                                     TGCAACCTGACCTTTGGCTCCTGGACCTACAATGGAAACCAGGTGGAC640                           CysAsnLeuThrPheGlySerTrpThrTyrAsnGlyAsnGlnValAsp                              170175180                                                                     ATATTCAATGCCCTGGACAGCGGTGACCTCTCTGACTTCATTGAAGAT688                           IlePheAsnAlaLeuAspSerGlyAspLeuSerAspPheIleGluAsp                              185190195200                                                                  GTGGAATGGGAGGTCCATGGCATGCCTGCTGTAAAGAACGTCATCTCC736                           ValGluTrpGluValHisGlyMetProAlaValLysAsnValIleSer                              205210215                                                                     TATGGCTGCTGCTCCGAGCCTTACCCAGATGTCACCTTCACTCTCCTT784                           TyrGlyCysCysSerGluProTyrProAspValThrPheThrLeuLeu                              220225230                                                                     CTGAAGAGGAGGTCCTCCTTCTACATCGTCAACCTCCTCATCCCTTGC832                           LeuLysArgArgSerSerPheTyrIleValAsnLeuLeuIleProCys                              235240245                                                                     GTCCTCATATCGTTCCTCGCTCCGTTGAGTTTCTATCTCCCAGCAGCC880                           ValLeuIleSerPheLeuAlaProLeuSerPheTyrLeuProAlaAla                              250255260                                                                     TCTGGGGAGAAGGTCTCTCTGGGAGTGACCATCCTATTGGCCATGACT928                           SerGlyGluLysValSerLeuGlyValThrIleLeuLeuAlaMetThr                              265270275280                                                                  GTGTTTCAGCTAATGGTGGCAGAGATCATGCCAGCCTCAGAAAATGTC976                           ValPheGlnLeuMetValAlaGluIleMetProAlaSerGluAsnVal                              285290295                                                                     CCTCTGATAGGAAAATACTACATAGCTACCATGGCCTTGATCACTGCC1024                          ProLeuIleGlyLysTyrTyrIleAlaThrMetAlaLeuIleThrAla                              300305310                                                                     TCCACAGCCCTTACCATCATGGTGATGAATATTCACTTCTGTGGAGCT1072                          SerThrAlaLeuThrIleMetValMetAsnIleHisPheCysGlyAla                              315320325                                                                     GAGGCACGGCCAGTGCCACACTGGGCCAAGGTGGTCATCCTGAAGTAC1120                          GluAlaArgProValProHisTrpAlaLysValValIleLeuLysTyr                              330335340                                                                     ATGTCCAGGATCTTGTTTGTCTACGATGTGGGTGAGAGCTGCCTTAGT1168                          MetSerArgIleLeuPheValTyrAspValGlyGluSerCysLeuSer                              345350355360                                                                  CCCCGCCACAGCCAGGAGCCAGAACAAGTCACGAAGGTTTATAGCAAA1216                          ProArgHisSerGlnGluProGluGlnValThrLysValTyrSerLys                              365370375                                                                     CTCCCAGAATCCAACCTGAAAACGTCCAGAAACAAAGACCTTTCCAGA1264                          LeuProGluSerAsnLeuLysThrSerArgAsnLysAspLeuSerArg                              380385390                                                                     AAGAAGGAAGTAAGAAAACTCTTAAAGAATGACCTGGGGTACCAGGGT1312                          LysLysGluValArgLysLeuLeuLysAsnAspLeuGlyTyrGlnGly                              395400405                                                                     GGGATCCCCCAGAATACTGACAGTTATTGTGCACGCTATGAAGCACTG1360                          GlyIleProGlnAsnThrAspSerTyrCysAlaArgTyrGluAlaLeu                              410415420                                                                     GCGAAAAATATCGAATACATTGCCAAGTGCCTCAAGGACCACAAGGCC1408                          AlaLysAsnIleGluTyrIleAlaLysCysLeuLysAspHisLysAla                              425430435440                                                                  ACCAACTCCAAGGGCAGCGAGTGGAAGAAGGTCGCCAAAGTCATAGAC1456                          ThrAsnSerLysGlySerGluTrpLysLysValAlaLysValIleAsp                              445450455                                                                     CGTTTCTTCATGTGGATTTTCTTTGCTATGGTGTTTGTCATGACCGTC1504                          ArgPhePheMetTrpIlePhePheAlaMetValPheValMetThrVal                              460465470                                                                     TTGATCATAGCAAGAGCAGATTAGCAGGAAAGAGGAGTGGGCTGGTAGGCA1555                       LeuIleIleAlaArgAlaAsp                                                         475                                                                           TTTAGAGATTTGGGGAAAACCCAATAAAATCACCTGAGATCTGCCCCAGCGTGTGAGTTC1615              AGCTGCTGTTCATACATAATTTAGGGGATAGGTTGCGTATGCTTTTATTCCTAACTTCAA1675              TCAATATCCTAGTTACATGTCAGGTTAAATCAAGCAGGAGATGCAAGGTTTCAAGGGTAA1735              AGGGCTGGAGGAAGAGAGTTAGAAAGGACCCTTTCACAGGCTCCCATGAAGGGGAGTGGT1795              GGCCTTCAGTTTATGTAATTATCTCTTTATTATTGTAGACAACAAAGCACAGTGTATTCC1855              TGCTTAAGATTTAAAGCAAGAAAAGACAAAACAAATTCATTCTCTTAGTCCTTAATAAAA1915              CTTTTTTTTTTAAACAAAAAAAA1938                                                   (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 479 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAsnArgProHisSerCysLeuSerPheCysTrpMetTyrPheAla                              151015                                                                        AlaSerGlyIleArgAlaValGluThrAlaAsnGlyLysTyrAlaGln                              202530                                                                        LysLeuPheSerAspLeuPheGluAspTyrSerSerAlaLeuArgPro                              354045                                                                        ValGluAspThrAspAlaValLeuAsnValThrLeuGlnValThrLeu                              505560                                                                        SerGlnIleLysAspMetAspGluArgAsnGlnIleLeuThrAlaTyr                              65707580                                                                      LeuTrpIleArgGlnThrTrpHisAspAlaTyrLeuThrTrpAspArg                              859095                                                                        AspGlnTyrAspArgLeuAspSerIleArgIleProSerAspLeuVal                              100105110                                                                     TrpArgProAspIleValLeuTyrAsnLysAlaAspAspGluSerSer                              115120125                                                                     GluProValAsnThrAsnValValLeuArgTyrAspGlyLeuIleThr                              130135140                                                                     TrpAspSerProAlaIleThrLysSerSerCysValValAspValThr                              145150155160                                                                  TyrPheProPheAspSerGlnGlnCysAsnLeuThrPheGlySerTrp                              165170175                                                                     ThrTyrAsnGlyAsnGlnValAspIlePheAsnAlaLeuAspSerGly                              180185190                                                                     AspLeuSerAspPheIleGluAspValGluTrpGluValHisGlyMet                              195200205                                                                     ProAlaValLysAsnValIleSerTyrGlyCysCysSerGluProTyr                              210215220                                                                     ProAspValThrPheThrLeuLeuLeuLysArgArgSerSerPheTyr                              225230235240                                                                  IleValAsnLeuLeuIleProCysValLeuIleSerPheLeuAlaPro                              245250255                                                                     LeuSerPheTyrLeuProAlaAlaSerGlyGluLysValSerLeuGly                              260265270                                                                     ValThrIleLeuLeuAlaMetThrValPheGlnLeuMetValAlaGlu                              275280285                                                                     IleMetProAlaSerGluAsnValProLeuIleGlyLysTyrTyrIle                              290295300                                                                     AlaThrMetAlaLeuIleThrAlaSerThrAlaLeuThrIleMetVal                              305310315320                                                                  MetAsnIleHisPheCysGlyAlaGluAlaArgProValProHisTrp                              325330335                                                                     AlaLysValValIleLeuLysTyrMetSerArgIleLeuPheValTyr                              340345350                                                                     AspValGlyGluSerCysLeuSerProArgHisSerGlnGluProGlu                              355360365                                                                     GlnValThrLysValTyrSerLysLeuProGluSerAsnLeuLysThr                              370375380                                                                     SerArgAsnLysAspLeuSerArgLysLysGluValArgLysLeuLeu                              385390395400                                                                  LysAsnAspLeuGlyTyrGlnGlyGlyIleProGlnAsnThrAspSer                              405410415                                                                     TyrCysAlaArgTyrGluAlaLeuAlaLysAsnIleGluTyrIleAla                              420425430                                                                     LysCysLeuLysAspHisLysAlaThrAsnSerLysGlySerGluTrp                              435440445                                                                     LysLysValAlaLysValIleAspArgPhePheMetTrpIlePhePhe                              450455460                                                                     AlaMetValPheValMetThrValLeuIleIleAlaArgAlaAsp                                 465470475                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 457 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetGluLeuSerThrValLeuLeuLeuLeuGlyLeuSerSerAlaGly                              151015                                                                        LeuValLeuGlySerGluHisGluThrArgLeuValAlaLysLeuPhe                              202530                                                                        GluAspTyrSerSerValValArgProValGluAspHisArgGluIle                              354045                                                                        ValGlnValThrValGlyLeuGlnLeuIleGlnLeuIleAsnValAsp                              505560                                                                        GluValAsnGlnIleValThrThrAsnValArgLeuLysGlnGlnTrp                              65707580                                                                      ValAspTyrAsnLeuLysTrpAsnProAspAspTyrGlyGlyValLys                              859095                                                                        LysIleHisIleProSerGluLysIleTrpArgProAspValValLeu                              100105110                                                                     TyrAsnAsnAlaAspGlyAspPheAlaIleValLysPheThrLysVal                              115120125                                                                     LeuLeuAspTyrThrGlyHisIleThrTrpThrProProAlaIlePhe                              130135140                                                                     LysSerTyrCysGluIleIleValThrHisPheProPheAspGluGln                              145150155160                                                                  AsnCysSerMetLysLeuGlyThrTrpThrTyrAspGlySerValVal                              165170175                                                                     AlaIleAsnProGluSerAspGlnProAspLeuSerAsnPheMetGlu                              180185190                                                                     SerGlyGluTrpValIleLysGluAlaArgGlyTrpLysHisTrpVal                              195200205                                                                     PheTyrSerCysCysProThrThrProTyrLeuAspIleThrTyrHis                              210215220                                                                     PheValMetGlnArgLeuProLeuTyrPheIleValAsnValIleIle                              225230235240                                                                  ProCysLeuLeuPheSerPheLeuThrSerLeuValPheTyrLeuPro                              245250255                                                                     ThrAspSerGlyGluLysMetThrLeuSerIleSerValLeuLeuSer                              260265270                                                                     LeuThrValPheLeuLeuValIleValGluLeuIleProSerThrSer                              275280285                                                                     SerAlaValProLeuIleGlyLysTyrMetLeuPheThrMetValPhe                              290295300                                                                     ValIleAlaSerIleIleIleThrValIleValIleAsnThrHisHis                              305310315320                                                                  ArgSerProSerThrHisIleMetProGluTrpValArgLysValPhe                              325330335                                                                     IleAspThrIleProAsnIleMetPhePheSerThrMetLysArgPro                              340345350                                                                     SerArgAspLysGlnGluLysArgIlePheThrGluAspIleAspIle                              355360365                                                                     SerAspIleSerGlyLysProGlyProProProMetGlyPheHisSer                              370375380                                                                     ProLeuIleLysHisProGluValLysSerAlaIleGluGlyValLys                              385390395400                                                                  TyrIleAlaGluThrMetLysSerAspGlnGluSerAsnAsnAlaAla                              405410415                                                                     GluGluTrpLysTyrValAlaMetValMetAspHisIleLeuLeuGly                              420425430                                                                     ValPheMetLeuValCysLeuIleGlyThrLeuAlaValPheAlaGly                              435440445                                                                     ArgLeuIleGluLeuHisGlnGlnGly                                                   450455                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 510 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetThrLeuSerHisSerAlaLeuGlnPheTrpThrHisLeuTyrLeu                              151015                                                                        TrpCysLeuLeuLeuValProAlaValLeuThrGlnGlnGlySerHis                              202530                                                                        ThrHisAlaGluAspArgLeuPheLysHisLeuPheGlyGlyTyrAsn                              354045                                                                        ArgTrpAlaArgProValProAsnThrSerAspValValIleValArg                              505560                                                                        PheGlyLeuSerIleAlaGlnLeuIleAspValAspGluLysAsnGln                              65707580                                                                      MetMetThrThrAsnValTrpLeuLysGlnGluTrpAsnAspTyrAsn                              859095                                                                        ValArgTrpAspProAlaGluPheGlyAsnValThrSerLeuArgVal                              100105110                                                                     ProSerGluMetIleTrpIleProAspIleValLeuTyrAsnAsnAla                              115120125                                                                     AspGlyGluPheAlaValThrHisMetThrLysAlaHisLeuPhePhe                              130135140                                                                     ThrGlyThrValHisTrpValProProAlaIleTyrLysSerSerCys                              145150155160                                                                  SerIleAspValThrPhePheProPheAspGlnGlnAsnCysLysMet                              165170175                                                                     LysPheGlySerTrpThrTyrAspLysAlaLysIleAspLeuGluGln                              180185190                                                                     MetGluArgThrValAspLeuLysAspTyrTrpGluSerGlyGluTrp                              195200205                                                                     AlaIleIleAsnAlaThrGlyThrTyrAsnSerLysLysTyrAspCys                              210215220                                                                     CysAlaGluIleTyrProAspValThrTyrTyrPheValIleArgArg                              225230235240                                                                  LeuProLeuPheTyrThrIleAsnLeuIleIleProCysLeuLeuIle                              245250255                                                                     SerCysLeuThrValLeuValPheTyrLeuProSerGluCysGlyGlu                              260265270                                                                     LysIleThrLeuCysIleSerValLeuLeuSerLeuThrValPheLeu                              275280285                                                                     LeuLeuIleThrGluIleIleProSerThrSerLeuValIleProLeu                              290295300                                                                     IleGlyGluTyrLeuLeuPheThrMetIlePheValThrLeuSerIle                              305310315320                                                                  ValIleThrValPheValLeuAsnValHisHisArgSerProSerThr                              325330335                                                                     HisAsnMetProAsnTrpValArgValAlaLeuLeuGlyArgValPro                              340345350                                                                     ArgTrpLeuMetMetAsnArgProLeuProProMetGluLeuHisGly                              355360365                                                                     SerProAspLeuLysLeuSerProSerTyrHisTrpLeuGluThrAsn                              370375380                                                                     MetAspAlaGlyGluArgGluGluThrGluGluGluGluGluGluAsp                              385390395400                                                                  GluAsnIleCysValCysAlaGlyLeuProAspSerSerMetGlyVal                              405410415                                                                     LeuTyrGlyHisGlyGlyLeuHisLeuArgAlaMetGluProGluThr                              420425430                                                                     LysThrProSerGlnAlaSerGluIleLeuLeuSerProGlnIleGln                              435440445                                                                     LysAlaLeuGluGlyValHisTyrIleAlaAspArgLeuArgSerGlu                              450455460                                                                     AspAlaAspSerSerValLysGluAspTrpLysTyrValAlaMetVal                              465470475480                                                                  ValAspArgIlePheLeuTrpLeuPheIleIleValCysPheLeuGly                              485490495                                                                     ThrIleGlyLeuPheLeuProProPheLeuAlaGlyMetIle                                    500505510                                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 497 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetGlyValValLeuLeuProProProLeuSerMetLeuMetLeuVal                              151015                                                                        LeuMetLeuLeuProAlaAlaSerGluAlaGluHisArgLeuPheGln                              202530                                                                        TyrLeuPheGluAspTyrAsnGluIleIleArgProValAlaAsnVal                              354045                                                                        SerHisProValIleIleGlnPheGluValSerMetSerGlnLeuVal                              505560                                                                        LysValAspGluValAsnGlnIleMetGluThrAsnLeuTrpLeuLys                              65707580                                                                      GlnIleTrpAsnAspTyrLysLeuLysTrpLysProSerAspTyrGln                              859095                                                                        GlyValGluPheMetArgValProAlaGluLysIleTrpLysProAsp                              100105110                                                                     IleValLeuTyrAsnAsnAlaAspGlyAspPheGlnValAspAspLys                              115120125                                                                     ThrLysAlaLeuLeuLysTyrThrGlyGluValThrTrpIleProPro                              130135140                                                                     AlaIlePheLysSerSerCysLysIleAspValThrTyrPheProPhe                              145150155160                                                                  AspTyrGlnAsnCysThrMetLysPheGlySerTrpSerTyrAspLys                              165170175                                                                     AlaLysIleAspLeuValLeuIleGlySerSerMetAsnLeuLysAsp                              180185190                                                                     TyrTrpGluSerGlyGluTrpAlaIleIleLysAlaProGlyTyrLys                              195200205                                                                     HisGluIleLysTyrAsnCysCysGluGluIleTyrGlnAspIleThr                              210215220                                                                     TyrSerLeuTyrIleArgArgLeuProLeuPheTyrThrIleAsnLeu                              225230235240                                                                  IleIleProCysLeuLeuIleSerPheLeuThrValLeuValPheTyr                              245250255                                                                     LeuProSerAspCysGlyGluLysValThrLeuCysIleSerValLeu                              260265270                                                                     LeuSerLeuThrValPheLeuLeuValIleThrGluThrIleProSer                              275280285                                                                     ThrSerLeuValIleProLeuIleGlyGluTyrLeuLeuPheThrMet                              290295300                                                                     IlePheValThrLeuSerIleValIleThrValPheValLeuAsnVal                              305310315320                                                                  HisTyrArgThrProThrThrHisThrMetProThrTrpValLysAla                              325330335                                                                     ValPheLeuAsnLeuLeuProArgValMetPheMetThrArgProThr                              340345350                                                                     SerGlyGluGlyAspThrProLysThrArgThrPheTyrGlyAlaGlu                              355360365                                                                     LeuSerAsnLeuAsnCysPheSerArgAlaAspSerLysSerCysLys                              370375380                                                                     GluGlyTyrProCysGlnAspGlyThrCysGlyTyrCysHisHisArg                              385390395400                                                                  ArgValLysIleSerAsnPheSerAlaAsnLeuThrArgSerSerSer                              405410415                                                                     SerGluSerValAsnAlaValLeuSerLeuSerAlaLeuSerProGlu                              420425430                                                                     IleLysGluAlaIleGlnSerValLysTyrIleAlaGluAsnMetLys                              435440445                                                                     AlaGlnAsnValAlaLysGluIleGlnAspAspTrpLysTyrValAla                              450455460                                                                     MetValIleAspArgIlePheLeuTrpValPheIleLeuValCysIle                              465470475480                                                                  LeuGlyThrAlaGlyLeuPheLeuGlnProLeuMetAlaArgAspAsp                              485490495                                                                     Thr                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 629 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetGluIleGlyGlyProGlyAlaProProProLeuLeuLeuLeuPro                              151015                                                                        LeuLeuLeuLeuLeuGlyThrGlyLeuLeuProAlaSerSerHisIle                              202530                                                                        GluThrArgAlaHisAlaGluGluArgLeuLeuLysArgLeuPheSer                              354045                                                                        GlyTyrAsnLysTrpSerArgProValGlyAsnIleSerAspValVal                              505560                                                                        LeuValArgPheGlyLeuSerIleAlaGlnLeuIleAspValAspGlu                              65707580                                                                      LysAsnGlnMetMetThrThrAsnValTrpValLysGlnGluTrpHis                              859095                                                                        AspTyrLysLeuArgTrpAspProGlyAspTyrGluAsnValThrSer                              100105110                                                                     IleArgIleProSerGluLeuIleTrpArgProAspIleValLeuTyr                              115120125                                                                     AsnAsnAlaAspGlyAspPheAlaValThrHisLeuThrLysAlaHis                              130135140                                                                     LeuPheTyrAspGlyArgValGlnTrpThrProProAlaIleTyrLys                              145150155160                                                                  SerSerCysSerIleAspValThrPhePheProPheAspGlnGlnAsn                              165170175                                                                     CysThrMetLysPheGlySerTrpThrTyrAspLysAlaLysIleAsp                              180185190                                                                     LeuValSerIleHisSerArgValAspGlnLeuAspPheTrpGluSer                              195200205                                                                     GlyGluTrpValIleValAspAlaValGlyThrTyrAsnThrArgLys                              210215220                                                                     TyrGluCysCysAlaGluIleTyrProAspIleThrTyrAlaPheIle                              225230235240                                                                  IleArgArgLeuProLeuPheTyrThrIleAsnLeuIleIleProCys                              245250255                                                                     LeuLeuIleSerCysLeuThrValLeuValPheTyrLeuProSerGlu                              260265270                                                                     CysGlyGluLysValThrCysAlaSerSerValLeuLeuSerLeuThr                              275280285                                                                     ValPheLeuLeuLeuIleThrGluIleIleProSerThrSerLeuVal                              290295300                                                                     IleProLeuIleGlyGluTyrLeuLeuPheThrMetIlePheValThr                              305310315320                                                                  LeuSerIleValIleThrValPheValLeuAsnValHisHisArgSer                              325330335                                                                     ProArgThrHisThrMetProAlaTrpValArgArgValPheLeuAsp                              340345350                                                                     IleValProArgLeuLeuPheMetLysArgProSerValValLysAsp                              355360365                                                                     AsnCysArgArgLeuIleGluSerMetHisLysMetAlaAsnAlaPro                              370375380                                                                     ArgPheTrpProGluProValGlyGluProGlyIleLeuSerAspIle                              385390395400                                                                  CysAsnGlnGlyLeuSerProAlaProThrPheCysAsnProThrAsp                              405410415                                                                     ThrAlaValGluThrGlnProThrCysArgSerProProLeuGluVal                              420425430                                                                     ProAspLeuLysThrSerGluValGluLysAlaSerProCysProSer                              435440445                                                                     ProGlySerCysProProProLysSerSerSerGlyAlaProMetLeu                              450455460                                                                     IleLysAlaArgSerLeuSerValGlnHisValProSerSerGlnGlu                              465470475480                                                                  AlaAlaGluAspGlyIleArgCysArgSerArgSerIleGlnTyrCys                              485490495                                                                     ValSerGlnAspGlyAlaAlaSerLeuAlaAspSerLysProThrSer                              500505510                                                                     SerProThrSerLeuLysAlaArgProSerGlnLeuProValSerAsp                              515520525                                                                     GlnAlaSerProCysLysCysThrCysLysGluProSerProValSer                              530535540                                                                     ProValThrValLeuLysAlaGlyGlyThrLysAlaProProGlnHis                              545550555560                                                                  LeuProLeuSerProAlaLeuThrArgAlaValGluGlyValGlnTyr                              565570575                                                                     IleAlaAspHisLeuLysAlaGluAspThrAspPheSerValLysGlu                              580585590                                                                     AspTrpLysTyrValAlaMetValIleAspArgIlePheLeuTrpMet                              595600605                                                                     PheIleIleValCysLeuLeuGlyThrValGlyLeuPheLeuProPro                              610615620                                                                     TrpLeuAlaAlaCys                                                               625                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 502 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetCysGlyGlyArgGlyGlyIleTrpLeuAlaLeuAlaAlaAlaLeu                              151015                                                                        LeuHisValSerLeuGlnGlyGluPheGlnArgArgLeuTyrLysGlu                              202530                                                                        LeuValLysAsnTyrAsnProLeuGluArgProValAlaAsnAspSer                              354045                                                                        GlnProLeuThrValTyrPheSerLeuSerLeuLeuGlnIleMetAsp                              505560                                                                        ValAspGluLysAsnGlnValLeuThrThrAsnIleTrpLeuGlnMet                              65707580                                                                      SerTrpThrAspHisTyrLeuGlnTrpAsnMetSerGluTyrProGly                              859095                                                                        ValLysAsnValArgPheProAspGlyGlnIleTrpLysProAspIle                              100105110                                                                     LeuLeuTyrAsnSerAlaAspGluArgPheAspAlaThrPheHisThr                              115120125                                                                     AsnValLeuValAsnAlaSerGlyHisCysGlnTyrLeuProProGly                              130135140                                                                     IlePheLysSerSerCysTyrIleAspValArgTrpPheProPheAsp                              145150155160                                                                  ValGlnGlnCysLysLeuLysPheGlySerTrpSerTyrGlyGlyTrp                              165170175                                                                     SerLeuAspLeuGlnMetGlnGluAlaAspIleSerSerTyrIlePro                              180185190                                                                     AsnGlyGluTrpAspLeuMetGlyIleProGlyLysArgAsnGluLys                              195200205                                                                     PheTyrGluCysCysLysGluProTyrProAspValThrTyrThrVal                              210215220                                                                     ThrMetArgArgArgThrLeuTyrTyrGlyLeuAsnLeuLeuIlePro                              225230235240                                                                  CysValLeuIleSerAlaLeuAlaLeuLeuValPheLeuLeuProAla                              245250255                                                                     AspSerGlyGluLysIleSerLeuGlyIleThrValLeuLeuSerLeu                              260265270                                                                     ThrValPheMetLeuLeuValAlaGluIleMetProAlaThrSerAsp                              275280285                                                                     SerValProLeuIleAlaGlnTyrPheAlaSerThrMetIleIleVal                              290295300                                                                     GlyLeuSerValValValThrValIleValLeuArgTyrHisHisHis                              305310315320                                                                  AspProAspGlyGlyLysMetProLysTrpThrArgIleIleLeuLeu                              325330335                                                                     AsnTrpCysAlaTrpPheLeuArgMetLysArgProGlyGluAspLys                              340345350                                                                     ValArgProAlaCysGlnHisLysProArgProCysSerLeuAlaSer                              355360365                                                                     ValGluLeuSerAlaGlyAlaGlyProProThrSerAsnGlyAsnLeu                              370375380                                                                     LeuTyrIleGlyPheArgGlyLeuGluGlyMetHisCysAlaProThr                              385390395400                                                                  ProAspSerGlyValValCysGlyArgLeuAlaCysSerProThrHis                              405410415                                                                     AspGluHisLeuMetHisGlyAlaHisProSerAspGlyAspProAsp                              420425430                                                                     LeuAlaLysIleLeuGluGluValArgTyrIleAlaAsnArgPheArg                              435440445                                                                     CysGlnAspGluSerGluValIleCysSerGluTrpLysPheAlaAla                              450455460                                                                     CysValValAspArgLeuCysLeuMetAlaPheSerValPheThrIle                              465470475480                                                                  IleCysThrIleGlyIleLeuMetSerAlaProAsnPheValGluAla                              485490495                                                                     ValSerLysAspPheAla                                                            500                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 511 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetLeuThrGluLysCysLeuGlyPhePheTyrSerGlyLeuCysLeu                              151015                                                                        TrpAlaSerLeuPheLeuSerPhePheLysValSerGlnGlnGlyGlu                              202530                                                                        SerGlnArgArgLeuTyrArgAspLeuLeuArgAsnTyrAsnArgLeu                              354045                                                                        GluArgProValMetAsnAspSerGlnProIleValValGluLeuGln                              505560                                                                        LeuSerLeuLeuGlnIleIleAspValAspGluLysAsnGlnValLeu                              65707580                                                                      IleThrAsnAlaTrpLeuGlnMetTyrTrpValAspIleTyrLeuSer                              859095                                                                        TrpAspGlnTyrGluTyrProGlyValGlnAsnLeuArgPheProSer                              100105110                                                                     AspGlnIleTrpValProAspIleLeuLeuTyrAsnSerAlaAspGlu                              115120125                                                                     ArgPheAspAlaThrPheHisThrAsnValLeuValAsnTyrSerGly                              130135140                                                                     SerCysGlnTyrIleProProGlyIleLeuLysSerThrCysTyrIle                              145150155160                                                                  AspValArgTrpPheProPheAspValGlnLysCysAspLeuLysPhe                              165170175                                                                     GlySerTrpThrHisSerGlyTrpLeuIleAspLeuGlnMetLeuGlu                              180185190                                                                     AlaAspIleSerAsnTyrIleSerAsnGlyGluTrpAspLeuValGly                              195200205                                                                     ValProGlyLysArgAsnGluLeuTyrTyrGluCysCysLysGluPro                              210215220                                                                     TyrProAspValThrTyrThrIleThrMetArgArgArgThrLeuTyr                              225230235240                                                                  TyrGlyLeuAsnLeuLeuIleProCysValLeuIleSerGlyLeuAla                              245250255                                                                     LeuLeuValPheLeuLeuProAlaAspSerGlyGluLysIleSerLeu                              260265270                                                                     GlyIleThrValLeuLeuSerLeuThrValPheMetLeuLeuValAla                              275280285                                                                     GluIleMetProAlaThrSerAspSerValProLeuIleAlaGlnTyr                              290295300                                                                     PheAlaSerIleMetValIleValGlyLeuSerValValValThrVal                              305310315320                                                                  LeuValLeuGlnPheHisHisHisAspProGlnAlaGlyLysMetPro                              325330335                                                                     ArgTrpValArgValIleLeuLeuAsnTrpCysAlaTrpPheLeuArg                              340345350                                                                     MetLysLysProGlyGluAsnIleLysProLeuSerCysLysTyrSer                              355360365                                                                     TyrProLysHisHisProSerLeuLysAsnThrGluMetAsnValLeu                              370375380                                                                     ProGlyHisGlnProSerAsnGlyAsnMetIleTyrSerTyrHisThr                              385390395400                                                                  MetGluAsnProCysCysProGlnAsnAsnAspLeuGlySerLysSer                              405410415                                                                     GlyLysIleThrCysProLeuSerGluAspAsnGluHisValGlnLys                              420425430                                                                     LysAlaLeuMetAspThrIleProValIleValLysIleLeuGluGlu                              435440445                                                                     ValGlnPheIleAlaMetArgPheArgLysGlnAspGluGlyGluGlu                              450455460                                                                     IleCysSerGluTrpLysPheAlaAlaAlaValIleAspArgLeuCys                              465470475480                                                                  LeuValAlaPheThrLeuPheAlaIleIleCysThrPheThrIleLeu                              485490495                                                                     MetSerAlaProAsnPheIleGluAlaValSerLysAspPheThr                                 500505510                                                                     __________________________________________________________________________

What is claimed is:
 1. Isolated nucleic acid encoding an alpha9nicotinic acetylcholine receptor (nAChR) subunit.
 2. Isolated nucleicacid according to claim 1, wherein said nucleic acid is DNA.
 3. DNAaccording to claim 2, wherein said DNA is a cDNA.
 4. DNA according toclaim 2, wherein said DNA encodes the amino acid sequence set forth inSEQ ID NO:2.
 5. DNA according to claim 2, wherein said DNA hybridizesunder high stringency conditions to the nucleotide sequence set forth inSEQ ID NO:1.
 6. DNA according to claim 2, wherein the nucleotidesequence of said DNA is at least 90% identical to the nucleotidesequence set forth in SEQ ID NO:1.
 7. A vector comprising DNA accordingto claim
 2. 8. A host cell containing a vector according to claim
 7. 9.A host cell according to claim 8, wherein said cell expresses functionalnACh receptor comprising at least one alpha9 nAChR subunit.
 10. A hostcell according to claim 9, wherein said receptor is homomeric.
 11. Ahost cell according to claim 9, wherein said receptor is heteromeric.12. Isolated mRNA complementary to DNA according to claim
 2. 13. Anucleic acid probe comprising at least 14 contiguous nucleotidesaccording to the nucleotide sequence set forth in SEQ ID NO:1.